Morphogen cell surface receptor

ABSTRACT

Disclosed are (1) nucleic acid sequences, amino acid sequences, homologies, structural features and various other data characterizing a morphogen cell surface receptor; (2) methods for producing receptor proteins, including fragments thereof, using recombinant DNA technology; (3) methods for identifying novel morphogen receptors and their encoding DNAs; (4) methods for identifying compounds capable of modulating endogenous morphogen receptor levels; and (5) methods for identifying morphogen receptor binding analogs useful in the design of morphogen agonists and antagonists for therapeutic, diagnostic and experimental uses.

REFERENCE TO RELATED APPLICATIONS

This application is a file-wrapper continuation of U.S. Ser. No.08/073,199 filed Jun. 7, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of tissue morphogenesisand more particularly to novel sequences encoding morphogen cell surfacereceptors.

BACKGROUND OF THE INVENTION

Cell differentiation is the central characteristic of tissuemorphogenesis which initiates during embryogenesis, and continues tovarious degrees throughout the life of an organism in adult tissuerepair and regeneration mechanisms. The degree of morphogenesis in adulttissue varies among different tissues and is related, among otherthings, to the degree of cell turnover in a given tissue.

The cellular and molecular events which govern the stimulus fordifferentiation of cells is an area of intensive research. In themedical and veterinary fields, it is anticipated that the discovery ofthe factor or factors which control cell differentiation and tissuemorphogenesis will advance significantly medicine's ability to repairand regenerate diseased or damaged mammalian tissues and organs.Particularly useful areas for human and veterinary therapeutics includereconstructive surgery and in the treatment of tissue degenerativediseases including arthritis, emphysema, osteoporosis, cardiomyopathy,cirrhosis, degenerative nerve diseases, inflammatory diseases, andcancer, and in the regeneration of tissues, organs and limbs. Innon-mammalian systems, such as insect systems, the discovery of suchfactors provide the basis for developing potent insecticides by, forexample, identifying compounds which inhibit or otherwise interfere withthe morphogenetic effect of these factors in the insect. (In this andrelated applications, the terms "morphogenetic" and "morphogenic" areused interchangeably.)

A number of different factors have been isolated in recent years whichappear to play a role in cell differentiation. Recently, various membersof the structurally related proteins of the transforming growth factor-β(TGF-β) superfamily of proteins have been identified as true tissuemorphogens.

This "family" of proteins, constituting a distinct subfamily within theTGF-β superfamily of structurally related proteins, share substantialamino acid sequence homology within their morphogenically activeC-terminal domains, including a conserved six or seven cysteineskeleton, and are capable of inducing tissue-specific morphogenesis in avariety of organs and tissues. The proteins apparently contact andinteract with progenitor cells e.g., by binding suitable cell surfacemolecules, predisposing or otherwise stimulating the cells toproliferate and differentiate in a morphogenically permissiveenvironment. The morphogens are capable of inducing the developmentalcascade of cellular and molecular events that culminate in the formationof new organ-specific tissue, including any vascularization, connectivetissue formation, and nerve innervation as required by the naturallyoccurring tissue.

A number of proteins useful in tissue morphogenesis have been identifiedto date, including proteins originally identified as bone inductiveproteins, such as the OP-1, (also referred to in related applications as"OP1"), OP-2 (also referred to in related applications as "OP2"), OP-3and the CBMP2 proteins, as well as amino acid sequence-related proteinssuch as BMP5, BMP6 and its murine homolog, Vgr-1, dpp and 60A (fromDrosophila), Vgl (from Xenopus), and GDF-1 (from mouse.) See, forexample, PCT documents US92/01968 and US92/07358 (published as WO92/15323 and WO 93/04692, respectively), the disclosures of which areincorporated herein by reference. These TGF-β superfamily memberscomprise a distinct subfamily of proteins different from other membersof the TGF-β superfamily in that the family of morphogenic proteins areable to induce the full cascade of events that result in formation offunctional tissue rather than merely inducing formation of fibrotic(scar) tissue as, for example, TGF-β does in many cases. Specifically,members of the morphogen family of proteins are capable of all of thefollowing in a morphogenically permissive environment: stimulating cellproliferation and cell differentiation, and supporting the growth andmaintenance of differentiated cells. The morphogenic proteins apparentlymay act as endocrine, paracrine or autocrine factors.

These proteins are capable of significant species "crosstalk." Forexample, dpp and 60A, two Drosophila proteins, can induce endochondralbone formation at a non-bony site in a standard rat bone formationassay. In their native form, however, the proteins appear to betissue-specific, each protein expressed in or provided to one or only afew tissues or, alternatively, expressed only at particular times duringdevelopment. For example, OP-1 is expressed and/or present primarily intissues of urogenital origin or bone tissue, although it has beenidentified in mammary, salivary gland tissue, and reproductive tissues,as well as in gastrointestinal tract tissue. GDF-1 appears to beexpressed primarily in neural tissue, while OP-2 appears to be expressedin early (e.g., 8-day) mouse embryo. The endogenous morphogens may besynthesized by the cells on which they act, by neighboring cells, or bycells of a distant tissue, the secreted protein being transported to thecells to be acted on.

Recently, the genetic sequences encoding receptors to several members ofthe TGF-β protein superfamily have been described. Lin et al. (1992)Cell 68:775-785, disclose the expression cloning of the TGF-β Type IIreceptor. Several groups have described genetic sequences encodingvarious activin (Type II) receptors in different species, includingmouse, rat, xenopus, and human. The overall amino acid sequence homologybetween these activin receptors is 50-80%. See, Matthews et al. (1991)Cell 65:973-982 and international patent application WO 92/20793,published Nov. 26, 1992, disclosing the "ActR II" sequence; Attisano etal., (1992) Cell 68:97-108, disclosing the "ActR-IIB" sequence; andLegerski et al. (1992) Biochem Biophys. Res.Comm'n 183:672-679. By aminoacid sequence homology to the TGF-β and activin Type II receptorsequences, the daf-1 gene, (Georgi et al. (1990) Cell 61:635-645),identified in C. elegans and having no known ligand to date, also isbelieved to encode a receptor for a TGF-β superfamily protein member.These disclosed receptors for TGF-β and activin are distinct from cellsurface receptors capable of specific binding interaction with themorphogens described herein, and do not bind these morphogenssignificantly (see, for example, Legerskie et al. (1992) Biochem.Biophys.Res. Comm'n. 183:672-679 and Attisano et al., (1992) Cell68:97-108.)

To date, the molecule or molecules with which the morphogens describedherein interact on the cell surface have not yet been identified.Identification of these cell surface molecules, with which themorphogens interact and through which they may mediate their biologicaleffect, is anticipated to significantly enhance elucidation of themolecular mechanism of tissue morphogenesis and to enable development ofmorphogen receptor binding "analogs", e.g., compounds (which may or maynot be amino acid-based macromolecules) capable of mimicking the bindingaffinity of a morphogen for its receptor sufficiently to act either as areceptor binding agonist or antagonist. These "analogs" have particularutility in therapeutic, diagnostic and experimental researchapplications.

It is an object of this invention to provide nucleic acid and amino acidsequences encoding a morphogen binding cell surface receptor, includingallelic, species, chimeric and mutant variants thereof. Another objectis to provide methods for identifying genes in a variety of speciesand/or tissues, and in a variety of nucleic acid libraries encodingmorphogen receptors which share little or no substantial amino acididentity with the extracellular domains of known TGF-β or activinreceptor molecules. Still another object is to provide means for theexpression of morphogen receptors, including truncated forms thereof,using recombinant DNA technology. Yet another object is to provide meansfor designing biosynthetic morphogen receptor-binding ligands and/or foridentifying natural-occurring ligands, including morphogen agonists andantagonists, using the morphogen receptor molecules of this invention,and analogs thereof. Still another object is to provides means andcompositions for modulating the endogenous expression or concentrationof these receptor molecules. Yet another object is to providecompositions and methods for creating useful insecticides. These andother objects and features of the invention will be apparent from thedescription, drawings and claims which follow.

SUMMARY OF THE INVENTION

A novel genetic sequence encoding a novel polypeptide chain, referred toherein as MR-1 (morphogen receptor-1) now has been discovered.Accordingly, the invention provides novel, substantially pure nucleicacid and amino acid sequences encoding the morphogen receptor, means forproducing morphogen receptor polypeptide chains using recombinant DNAtechnology, means for identifying sequences encoding other morphogenreceptors ("MR"), and means for designing morphogen receptor bindinganalogs, referred to herein as morphogen analogs.

The morphogen receptors of this invention share a conserved structure,including an extracellular domain generally composed of about 120-150amino acids, a transmembrane domain sufficient to span a cellularmembrane one time, and an intracellular (cytoplasmic) domain havingserine/threonine kinase activity. The morphogen receptors of thisinvention share little or no substantial amino acid sequence identity(e.g., less than 30%) with the extracellular ligand binding domains ofknown Type II TGF-β or activin receptor molecules. In a preferred aspectof the invention, morphogen receptors share less than 26% amino acidsequence identity with the extracellular ligand binding domains of knownTGF-β or activin receptor molecules.

As used herein, "morphogen analog" refers to any molecule capable ofmimicking the binding activity of a morphogen sufficiently to act as amorphogen receptor binding ligand. These analog ligands may act asmorphogen agonists capable of mimicking the morphogen both in receptorbinding and in inducing a transmembrane effect. Alternatively, theanalog ligands may act as morphogen antagonists, capable of mimickingthe morphogen in receptor binding, thereby blocking the naturalmorphogen from interacting with its receptor, but without inducing atransmembrane effect, for example. Morphogen analogs may be aminoacid-based, or may be composed of other chemical structures, and may benaturally sourced or synthetically produced.

As used herein, "amino acid sequence homology" is understood to meanamino acid sequence similarity, and homologous sequences share identicalor similar amino acids, where similar amino acids are conserved aminoacids as defined by Dayoff et al., Atlas of Protein Sequence andStructure; vol.5, Suppl.3, pp.345-362 (M. O. Dayoff, ed., Nat'l BioMed.Research Fdn., Washington D.C. 1978.) Thus, a candidate sequence sharing60% amino acid homology with a reference sequence requires that,following alignment of the candidate sequence with the referencesequence, 60% of the amino acids in the candidate sequence are identicalto the corresponding amino acid in the reference sequence, or constitutea conserved amino acid change thereto. "Amino acid sequence identity" isunderstood to require identical amino acids between two alignedsequences. Thus, a candidate sequence sharing 60% amino acid identitywith a reference sequence requires that, following alignment of thecandidate sequence with the reference sequence, 60% of the amino acidsin the candidate sequence are identical to the corresponding amino acidin the reference sequence.

As used herein, all homologies and identities calculated use MR-1 as thereference sequence, with the extracellular domain reference sequenceconstituting residues 25-147 of Seq. ID No.1; and the intracellulardomain reference sequence constituting residues 209-492 of Seq. ID No.1.Also as used herein, sequences are aligned for homology and identitycalculations as follows: Sequences are aligned by eye to maximizesequence identity. Where amino acid extracellular domain sequences arecompared, the alignment first maximizes alignment of the cysteinespresent in the two sequences, then modifies the alignment as necessaryto maximize amino acid identity and similarity between the twosequences. Where amino acid intracellular domain sequences are compared,sequences are aligned to maximize alignment of conserved amino acids inthe kinase domain, where conserved amino acids are those identified byboxes in FIG. 3. The alignment then is modified as necessary to maximizeamino acid identity and similarity. In all cases, internal gaps andamino acid insertions in the candidate sequence as aligned are ignoredwhen making the homology/identity calculation. Exemplary alignments areillustrated in FIGS. 2 and 3 where the amino acid sequences for theextracellular and intracellular domains, respectively are presented insingle letter format. In the figures "gaps" created by sequencealignment are indicated by dashes. See, for example, in FIG. 3, the fourdashes occurring after "CEHF" at position 39 in the MR-1 sequence. Ofcourse, the next amino acid in the MR-1 sequence is "D", aspartic acid,which appears following the dashes, at position 40.

Also as used herein, "mutant variant" is understood to mean an aminoacid variant form of the receptor molecule wherein the amino acidchanges in the sequences do not alter significantly the activity (e.g.,morphogen binding or kinase activity) of the receptor molecule, and thevariant molecule performs substantially the same function insubstantially the same way as the naturally occurring form of themolecule. These variants also may be naturally occurring or may bebiosynthetically constructed by using standard recombinant DNAtechniques or chemical protein synthesis methodologies. In a preferredembodiment, these mutant variants having a binding affinity for amorphogen ligand with a Kd less than about 10⁻⁷ M (see below.)

Finally, as used herein, ligand-receptor binding specificity isunderstood to mean a specific, saturable noncovalent interaction betweenthe ligand and the receptor, and which is subject to competitiveinhibition by a suitable competitor molecule. Preferred bindingaffinities (defined as the amount of ligand required to fill one-half(50%) of available receptor binding sites) are described herein bydissociation constant (Kd). Preferred binding affinities of ligandshaving specificity for a given receptor molecule of the invention have aKd of less than 10⁻⁷ M, preferably less than 10⁻⁸ M. In anotherpreferred embodiment, the receptor molecules have little or nosubstantial binding affinity for TGF-β or activin. That is, TGF-β andactivin (or inhibin) have a binding affinity for the morphogen receptorsof this invention with a Kd greater than 10⁻⁷ M.

Useful morphogen receptor polypeptide chains include those encoded bythe DNA sequence of SEQ ID NO. 1, including allelic and species variantsthereof, as well as other naturally occurring and biosynthetic mutants,including truncated forms thereof.

In one aspect, the morphogen receptor polypeptide chains of thisinvention comprise: (1) an extracellular domain defining athree-dimensional conformation capable of binding a morphogen withspecificity, (2) a transmembrane domain sufficient to span a cellularmembrane, and (3) an intracellular (cytoplasmic) domain that isactivated in response to the binding interaction of the extracellulardomain with a morphogen or a morphogen analog.

In a preferred embodiment, the morphogen receptor comprises apolypeptide chain having a sequence of about 500 amino acids,glycosylated under naturally-occurring conditions, and comprising amolecular weight of about 58-90 kDa. As used herein, all molecularweights are apparent molecular weights, determined by comparison tomolecular weight standards on a standard SDS-polyacrylamideelectrophoresis gel.

In another embodiment, the morphogen receptor comprises anunglycosylated polypeptide chain having a sequence of at least about 120amino acids and a molecular weight of about 15 kDa, or of about 500amino acids and a molecular weight of about 59 kDa.

In another aspect, the polypeptide chains of this invention includetruncated forms of the sequence of SEQ ID NO. 1, including allelic,species and mutant variants thereof, comprising the extracellulardomain, or a portion thereof sufficient to bind a morphogen or morphogenanalog with specificity. A currently preferred truncated form includespolypeptide chains comprising an amino acid sequence having less thanabout 200 amino acids and comprising part or all of the sequence definedby residues 25-147 of SEQ ID NO. 1.

In another aspect of the invention, the polypeptide chains of thisinvention include a truncated form of the sequence of ID NO. 1,including allelic, species and mutant variants thereof, comprising atleast the cytoplasmic domain, and preferably part or all of thetransmembrane domain as well. A currently preferred "intracellular"truncated form includes polypeptide chains comprising part or all of thesequences defined by residues 209-492 of Seq. ID No. 1, or residues173-492 therein, or residues 148-492 therein.

In still another aspect of the invention, the polypeptide chains of thisinvention include a truncated form of the sequence of ID NO. 1,including allelic, species and mutant variants thereof, comprising atleast the extracellular or cytoplasmic domain or a fragment thereof,linked to a peptide sequence useful for anchoring the polypeptide chainto a surface. Such anchors have particular utility for purification ofthe protein. A currently preferred anchor sequence particularly usefulfor purification protocols is a sequence comprising six histidines,referred to herein and in the art as a "hexa-His" or (His)₆ sequence.Another preferred anchor sequence useful for intracellular truncatedforms includes peptide sequences, e.g., 5-15 amino acids in length,preferably derived from the C-terminal end of the extracellular domain.

In still another aspect of the invention, the polypeptide chains of thisinvention include chimeric receptor molecules comprising both anextracellular and intracellular (cytoplasmic) domain, and wherein atleast either the extracellular domain or the cytoplasmic domain isencoded by the appropriate subpart of the sequence of SEQ ID NO. 1,including allelic, species and mutant variants thereof. In still anotheraspect, the extracellular or intracellular domains themselves mayconstitute chimeric sequences comprising the appropriate subpart of thesequence of Seq. ID NO.1.

In another aspect, the invention comprises receptor molecules having anextracellular ligand binding domain having less than 30% amino acididentity, and preferably less than 20% amino acid identity with theligand binding domain of activin Type II receptors, or TGF-β Type IIreceptors currently known in the art.

In another aspect, the invention comprises morphogen receptor moleculescapable of specific binding interaction with a morphogen. In onepreferred embodiment, the morphogen comprises the amino acid sequence ofthe drosophila proteins DPP or 60A. In another preferred embodiment, themorphogen comprises OP-1 or species, allelic and mutant variantsthereof. In another preferred embodiment the morphogen comprises theamino acid sequence of OP-2 or OP-3,Vgr-1, Vgl, BMP2-6, GDF-1, orGeneric Sequences 1-6, defined in PCT US92/01968 (published as WO92/15323).

Another aspect of the invention comprises morphogen receptor polypeptidechains capable of binding a morphogen with specificity, having little orno substantial amino acid identity with the extracellular domain ofknown TGF-β or activin receptors, and encoded by nucleic acid whichhybridize to part or all of the cytoplasmic domain of MR-1 (residues209-492) of SEQ ID NO. 1 under low stringent hybridization conditions.As used herein, low stringency hybridization conditions are understoodto be those defined in Maniatis et al. Molecular Cloning (A laboratoryManual), Cold Spring Harbor Laboratory 2nd Ed. (1989). Exemplaryconditions include: hybridization in 30% formamide, 5× SSPE, 5×Denhardt's Solution, and 0.5% SDS at 37° C. overnight, and washing in2.0× SSPE, 0.5% SDS at 37° C.

In another aspect, the encoding nucleic acids hybridize with a nucleicacid that encodes either the extracellular domain or the intracellulardomain under high stringency conditions, as defined in Maniatis et al.

In still another aspect, the invention comprises morphogen receptorpolypeptide chains capable of binding a morphogen or morphogen analogwith specificity and encoded by nucleic acids which hybridize with anucleic acid that encodes part or all of the extracellular domain ofSeq. ID No. 1 under low stringency conditions.

In another aspect, the invention comprises morphogen receptor moleculescapable of binding a morphogen with specificity but having littlebinding affinity for TGF-β or activin, and encoded by nucleic acids thatcan be amplified with any of the primer sequences described below (SEQID NOS. 4-7, respectively) in a standard PCR amplification protocol:

    ______________________________________                                        PRIMER #1: ATTSKWRCTY TTGADGTCSC KGTG (Seq. ID NO. 4)                         PRIMER# 2: ATYGCBCACM GSGAYHTCAA RAG (Seq. ID NO. 5)                          PRIMER #3: GAATCTGTSG CHGTSAARRT HTTYCC (Seq. ID NO. 6)                       PRIMER #4: TCCAGSACYT CNGGDGCCAK RTA (Seq. ID NO. 7).                         ______________________________________                                    

In one preferred embodiment, the primers are modified to reflect anon-drosophila codon bias, preferably a human codon bias.

In another aspect, the invention provides a method for identifyinggenetic sequences encoding human and other mammalian morphogen receptorsby first identifying sequences encoding these receptors in a drosophilaor other genome having less complexity than a mammalian genome andlikely to encode fewer members of the family of morphogen receptors, andusing the thus identified receptor sequences to locate their mammalianhomologs. Because of the significant species "cross talk" evidenced bymorphogens (e.g., ability of these proteins to be biologically activeacross different species, including forming the necessary interactionswith xenogenic morphogen receptors) it is anticipated that this approachto locating mammalian receptor sequences will be reliable and easeidentification of the genes encoding this family of receptors inmammals.

In another preferred embodiment, the morphogen receptor molecules haveless than 30% amino acid identity, preferably less than 26% identity, intheir extracellular domains with the extracellular domain of activin Aor activin B Type II receptors, or TGF-β Type II receptors.

In another aspect, the invention comprises molecules capable of bindinga morphogen with specificity and sharing at least 30% amino acidsequence identity with the sequence defined by residues 25-147 of Seq.ID No.1, preferably at least 40%, more preferably 50%, identity. Inanother aspect, the invention comprises receptor molecules comprisingboth an extracellular and intracellular domain, wherein the amino acidsequence of the receptor molecule shares at least 35% amino acidsequence identity, preferably at least 55%, and more preferably at least65% identity with the sequence defined by residues 210-490 of Seq. IDNo.1, and wherein the receptors have little or no binding affinity foractivin or TGF-β (e.g., binding affinity of these receptors for theseligands have a Kd greater than 10⁻⁷ M.)

In still another aspect, the invention provides molecules useful in thedesign and/or identification of morphogen analogs as described below.The molecules useful in these assays may include part or all of thesequence of SEQ ID NO. 1, including allelic, species and mutant variantsthereof. Currently preferred for these assays are molecules comprisingat least the sequence which defines the extracellular domain, e.g.,residues 25-147.The morphogen analogs identified in these assays may actas morphogen agonists or antagonists.

In one embodiment of the invention, useful morphogen analogs includeantibodies capable of specifically recognizing and binding to themorphogen binding surface of the receptor. These antibodies may bemonoclonal or polyclonal, or may be biosynthetic derivatives thereof,including, but not limited to, for example, monoclonal fragments, suchas single chain F_(v) fragments, referred to in the literature as sF_(v)s, BABs and SCAs, and chimeric monoclonals, in which portions of themonoclonals are humanized (excluding those portions involved in antigenrecognition (e.g., complementarity determining regions, "CDRs".) See,for example, U.S. Pat. Nos. 5,091,513, 5,132,405, and U.S. Ser No.955,399, soon to issue as U.S. Pat. No. 5,258,489. Biosyntheticchimeras, fragments and other antibody derivatives may be synthesizedusing standard recombinant DNA methodology and/or automated chemicalnucleic acid synthesis methodology well described in the art and asdescribed below.

In another embodiment of the invention, antibodies to the intracellulardomain are contemplated. These antibodies will have particular utilityin receptor purification schemes.

In still another aspect the invention comprises molecules useful inscreening assays to identify molecules that modulate endogenousmorphogen receptor concentrations. Useful assay methodologies may bemodeled on those described in PCT US92/07359 (published as WO 93/05172),and as described below.

In another embodiment, useful antagonists include soluble forms of thereceptor binding surface which are capable of competing for morphogenbinding at a target site. Useful applications for antagonists includetheir use as therapeutics to modulate uncontrolled differentiated tissuegrowth, such as malignant transformations as occurs, for example, inosteosarcomas or Paget's disease. Still other useful applicationsinclude their use as insecticides, where the antagonist is a moleculehaving specificity for a non-mammalian insect-specific morphogenreceptor and can inhibit or otherwise interfere with insect growth andtissue development.

The invention thus relates to compositions and methods for the use ofmorphogen receptor polypeptide chains in diagnostic, therapeutic andexperimental procedures. Active morphogen receptors useful in thecompositions and methods of this invention may include truncated or fulllength forms, as well as forms having varying glycosylation patterns.Active morphogen receptors of the invention also include chimericconstructs as described below, comprising both an MR-1 sequence and anon-MR-1 sequence. Active MR-1 can be expressed from intact or truncatedgenomic or cDNA, or from synthetic DNAs in prokaryotic or eukaryotichost cells, and purified, cleaved, refolded and oxidized as necessary toform active molecules. Useful host cells include prokaryotes, includingE. coli and B. subtilis, and eukaryotic cells, including mammaliancells, such as fibroblast 3T3 cells, CHO, COS, melanoma or BSC cells,Hela and other human cells, the insect/baculovirus system, as well asyeast and other microbial host cell systems.

Thus, in view of this disclosure, skilled genetic engineers can isolategenes from libraries of various different species which encodeappropriate amino acid sequences, or construct them fromoligonucleotides, and can express these genetic sequences in varioustypes of host cells to produce large quantities of morphogen receptorpolypeptide chains capable of binding morphogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the encoded MR-1 amino acidsequence, wherein the first and second stippled regions identify thesignal sequence and transmembrane domains, respectively; the first openbox identifies the extracellular binding ligand binding domain; thesecond open box identifies the intracellular serine/threonine kinasedomain; broken lines indicate the position of cysteine residues;diamonds indicate potential glycosylation sites, and the numbersappearing below the sequence indicate amino acid residues;

FIG. 2 is a homology alignment of the extracellular domains of MR-1 andother known receptors which bind various members of the TGF-βsuperfamily of structurally related proteins, aligned to maximize aminoacid identity, and wherein conserved amino acids are identified byboxes; and

FIG. 3 is a homology alignment of the intracellular domain of MR-1 andthe various known receptors for proteins in the TGF-β superfamily ofstructurally related proteins, aligned to maximize amino acid identity,and wherein conserved amino acids are boxed. Asterisks indicate theregions of high amino acid and/or nucleotide sequence homology on whichPCR primer sequences were based.

DETAILED DESCRIPTION

A cloning protocol has been developed which enables identification ofnucleic acid sequences encoding a morphogen cell surface receptor(MR-1). The protocol takes advantage of the significant "cross talk"among morphogen species variants, and relies on a PCR DNA amplificationprocedure as disclosed herein for identifying genetic sequences in aDrosophila genome and encoding candidate receptor molecules. DNAsencoding candidate morphogen receptor molecules are DNAs encodingmembrane spanning molecules having extracellular domains sharing lessthan 30% identity and preferably less than 26% identity with theextracellular domains of other, known receptor molecules whose ligandsare members of the TGF-β superfamily (e.g., the daf-1 receptor and theType II TGF-β and activin receptors.) The thus identified sequencesfound to encode morphogen receptors then can be used to identify theirmammalian, including human, homologs and related variants.

The Drosophila genome was chosen herein as the source genome as anexample of a genetically less complex genome likely to encode fewermembers of the family of morphogen receptors identified to date inmammals. Other genomes, such as that of Xenopus, for example, also maybe used to advantage. Because of the significant species "cross talk"evidenced by morphogens (e.g., ability of these proteins to bebiologically active across different species, including forming thenecessary interactions with xenogenic morphogen receptors) it isanticipated that this approach to locating mammalian receptor sequenceswill be reliable and ease identification of the genes encoding thisfamily of receptors.

The cloning protocol allows identification and recombinant expression ofa novel protein, MR-1, comprising an extracellular domain, atransmembrane region and a cytoplasmic domain, and which is capable ofbinding one or more morphogens with specificity. The protein has beencharacterized as follows: its nucleotide and encoded amino acidsequences have been determined, its potential glycosylation sites havebeen identified in the amino acid sequence, antibodies to the proteinhave been raised and tested, and portions of the protein have beenrecombinantly produced.

Identification of MR-1, including identification of its nucleic acidsequence, allows creation of probes to identify other morphogen receptor(MR) sequences in other species, as well as to detect the tissuedistribution of MR expression. The probes may be derived from MR-1,preferably derived from the extracellular domain, and modified, forexample, to account for a preferred species codon bias. Alternatively,the MR-1 sequence information may be used to refine the primer sequencesdescribed below, or to design other degenerate primer sequences foramplifying other DNAs. Under naturally occurring conditions, individualmorphogens are preferentially expressed in, or preferentially act on,different tissues. Accordingly, it is anticipated that a family ofmorphogen receptors exist that are expressed preferentially in differenttissues and which bind preferentially with preferred members of themorphogen family of proteins.

The morphogen receptor disclosed herein, MR-1, including allelic,species and mutant variants of MR-1, also can be used in protocols toidentify receptor binding ligands that function as morphogen analogs andwhich may be used as morphogen agonists or antagonists, for therapeutic,diagnostic and experimental uses as described herein below. Theseligands may be naturally occurring molecules identified in, for example,a high flux screen, or they may be designed and biosynthetically createdusing a rational drug design and established structure/functionanalysis. The ligands may be amino acid-based or may be composed ofnon-proteinaceous chemical structures. Useful ligands also may beantibodies, preferably monoclonal antibodies or synthetic derivativesthereof, such as monoclonal single chain F_(v) fragments known in theart as sF_(v) s, BABs, and SCAs (see below.) Moreover, soluble forms ofthe protein, e.g., forms consisting essentially of the extracellulardomain or a fragment thereof sufficient to bind a morphogen withspecificity, may be used as a soluble therapeutic morphogen antagonists,as described below.

The MR sequence identified herein also may be used to create chimeric MRsequences, wherein, for example, part or all of either the extracellulardomain or the intracellular domain is a non-MR sequence or is a sequencefrom another, non-MR-1 morphogen receptor sequence. These chimeric MRpolypeptide chains may be synthesized using standard recombinant DNAmethodology and/or automated chemical nucleic acid synthesis methodologywell described in the art and as disclosed below. Chimerics may beuseful, for example, in morphogen analog assays, wherein the MRextracellular binding domain is coupled to an intracellular domain thatis well characterized and/or readily detectable as a second messengerresponse system, as described below.

The cloning procedure for obtaining MR nucleic acid sequences, as wellas other material aspects concerning the nature and utility of thesesequences, including how to make and how to use the subject matterclaimed, will be further understood from the following, whichconstitutes the best mode currently contemplated for practicing theinvention.

EXAMPLE 1 IDENTIFICATION OF MR-1

A. Cloning

A morphogen receptor gene sequence was isolated by PCR amplification,using degenerate sequences as primers to amplify a DNA fragment ofDrosophila genomic DNA. The Drosophila genome, known to encode at leasttwo morphogens, dpp and 60A, was chosen as the initial probing librarybecause of its smaller size compared to that of higher organisms. ThePCR primer sequences were designed based on predictions from observedhomologies with the deduced amino acid sequences of known receptorsequences of other members of the TGF-β superfamily of proteins, using aDrosophila codon bias and creating degeneracies within the sequence asnecessary.

Specifically, genetic sequences encoding TGF-β Type II and activin TypeII receptor molecules are known, as is the daf-1 receptor from C.elegans (see Lin et al., (1992) Cell 68:775-785; Matthews et al, (1991)Cell65:973-982; Attisano et al., (1992) Cell 68:97-108; and Georgi etal., (1990) Cell 61:635-645, respectively, and in Seq. ID Nos. 8-11,respectively.) All four receptors share the same overall structure,having a relatively short extracellular domain, a transmembrane sequencesufficient to span the cellular membrane once, and an intracellularsequence that is believe to act as a serine/threonine kinase. While theextracellular domains of the TGF-β, activin and daf-1 receptors sharelittle amino acid homology or identity, the intracellular domains sharemore homology (between about 30-65% amino acid identity.) The variousactivin receptor species share between about 55-78% amino acid identityin their extracellular and intracellular domains.

TGF-β and activin, known ligands for two of the three receptors, alsoshare a similar overall structural motif with each other and withmorphogens, based on a conserved cysteine pattern within theirC-terminal domains. Several of the morphogenic activities characteristicof morphogens also are demonstrated by TGF-β and activin/inhibins.Accordingly, on the belief that the morphogen receptors also likelyshare a similar overall structural motif with the Type II TGF-β andactivin receptors, the intracellular serine/threonine kinase domains ofthe known receptors were aligned and regions of high amino acid homologyidentified for designing degenerate primer sequences. Four candidateregions were identified, indicated by asterisks in FIG. 3. The number inparentheses beneath these asterisks corresponds to the primer number.

Oligonucleotides were prepared with degeneracies (512 fold) introducedto include multiple probable sequences encoding this amino acidsequence, while maintaining the preferred Drosophila codon bias. Inaddition, where possible, observed conserved nucleotide sequences wereexploited. The four primers created (#1-4) are shown below and in Seq.ID Nos. 4-7, respectively:

    ______________________________________                                        Primer 1 (#3501):                                                                       ATT SKW RCT YTT GAD GTC SCK GTG                                     Primer 2 (#3502):                                                                       ATY GCB CAC MGS GAY HTC AAR AG                                      Primer 3 (#3503):                                                                       GAA TCT GTS GCH GTS AAR RTH TTY CC                                  Primer 4 (#3504):                                                                       TCC AGS ACY TCN GGD GCC AKR TA                                      ______________________________________                                    

Single Letter Code (IUPAC): ##STR1##

These primers then were used in a PCR reaction using Drosophila genomicDNA and standard procedures well known in the art, see for example,Saiki et al. (1985) Science 230:1350-1354.

Briefly, degenerate oligonucleotides were synthesized on a standardautomated DNA synthesizer, (e.g., Cruachem PS250, Scotland) followingmanufacturer's instructions, and then purified using standardprocedures.

PCR reactions were performed with a commercially available thermalcycler and reagent kit (e.g., GeneAmp, Perkins & Elmer Corp., Norwalk)and following the manufacturer's instructions in a standard PCRprotocol: in a 100 μl final volume with 1 μg drosophila genomic DNA, 1μM final concentration of each primer, 0.2 mM dNTP's, 50 mM KCl 10 mMTris-HCl(pH 9.0 at 25° C.) 0.1% Triton X-100, 1.5 mM MgCl₂, 2.5 units ofTaq polymerase. The reaction mixture was heated to 60° C. beforeaddition of the nucleotides and polymerase and amplification wasperformed for 40 cycles:

    ______________________________________                                        program:    94° C. 1 inin                                                          55° C. 1 min. 30 sec.                                                  72° C. 1 min. 20 sec. + 1 sec followed by a                single extension of                                                                       72° C. 5 min.                                                          4° C. hold                                                 ______________________________________                                    

Amplified products then were separated by standard polyacrylamide gelelectrophoresis, and fragments of the appropriate size (e.g., 474 bases)excised and purified using standard procedures. These fragments thenwere subcloned into a standard, commercially available cloning vector(e.g., available from Invitrogen Inc., San Diego) compatible with thePCR-generated DNA fragment ends. DNA from individual clones then wasprepared by standard alkaline lysis and the isolated DNAs sequencedusing standard procedures and commercially available reagents (e.g.,dideoxy sequencing, U.S. BioChem Sequencing Kit, Cleveland.)

PCR amplification with primers 3 and 4 identified a 470 base pairfragment, a partial sequence of which appears in Seq. ID No.3, andcorresponding to the nucleotide sequence encoding residues 224 to 382 ofSeq. ID No. 1. The 470 base pair fragment then was used to create aprobe sequence (by random priming) for standard DNA library screeningunder high stringency conditions (e.g., washed at 0.1× SSPE, 0.1% SDS,at 50° C.) to identify the complete coding sequence in a Drosophila cDNAlibrary (UniZAPXR Drosophila embryo cDNA library, Stratagene, La Jolla)and standard library screening procedures (see, for example, Maniatis,Molecular Cloning: A Laboratory Manual (2nd Ed'n, 1989, Cold SpringHarbor, N.Y.)

B. MR-1 Sequence Characterization.

The complete coding sequence for the MR-1 cDNA is described in SEQ IDNO. 1 and is represented schematically in FIG. 1. The full length DNAsequence encodes a polypeptide chain about 516 amino acids in lengththat is glycosylated under standard physiological conditions. In FIG. 1the first and second stippled regions represent the signal peptidesequence and the transmembrane domain, respectively. The signal peptidesequence, corresponding essentially to residues 1-24 of Seq. ID No.1, isapparently 24 amino acids in length, the cleavage site being determinedby the method of von Heijne (1986) Nucleic Acids Research14:4683-4691.)The transmembrane region, corresponding essentially to residues 148-172of Seq. ID No. 1, is rich in hydrophobic residues and is sufficient tospan the cellular membrane one time. The open region correspondingessentially to residues 25-147 of Seq. ID No. 1 corresponds to theextracellular domain of the protein. This region is rich in cysteines,represented in the figure by vertical hatched lines, indicating thatthis region of the polypeptide chain likely is highly disulfide bonded.This region, which constitutes the ligand binding domain, shares littleor no amino acid homology with the corresponding sequences of the TGFβ,activin or daf-1 receptor sequences (e.g., less than 26% amino acididentity.) Three possible glycosylation sites occur in this region, thepositions being indicated by diamonds in the figure. The crosshatchedregion, corresponding to residues 173-492 of Seq. ID No.1, correspondsto the intracellular (cytoplasmic domain) which shares 30-67% amino acididentity with the intracellular serine/threonine kinase domains of theother known serine/threonine kinase containing molecules (see FIG. 3.)

EXAMPLE 2 MR EXPRESSION

A. General Considerations

MR DNA, or a synthetic form thereof, can be inserted, using conventionaltechniques well described in the art (see, for example, Maniatis (1989)Molecular Cloning A Laboratory Manual), into any of a variety ofexpression vectors and transfected into an appropriate host cell toproduce recombinant MR, such as MR-1 polypeptide chains, including bothfull length and truncated forms thereof. Shortened sequences, forexample, can be used for the production of soluble receptor fragments.

Useful host cells include E. coli, Saccharomyces, the insect/baculoviruscell system, myeloma cells, and various other mammalian cells. The fulllength form of the proteins of this invention preferably are expressedin mammalian cells, as disclosed herein. Soluble forms may be expressedfrom both mammalian or bacterial cell systems. The vector additionallymay include various sequences to promote correct expression of therecombinant protein, including transcription promoter and terminationsequences, enhancer sequences, preferred ribosome binding sitesequences, preferred mRNA leader sequences, preferred protein processingsequences, preferred signal sequences for protein secretion, and thelike. The DNA sequence encoding the gene of interest also may bemanipulated to remove potentially inhibiting sequences or to minimizeunwanted secondary structure formation. The recombinant morphogenreceptor also may be expressed as a fusion protein. After beingtranslated, the protein may be purified from the cells themselves orrecovered from the culture medium. The DNA also may include sequencesthat aid in expression and/or purification of the recombinant protein.One useful sequence for example, is a hexa-His (His₆) sequence, whichadds a histidine tail to anchor the protein to an IMAC Cu2+ column (seebelow.)

For example, the DNA encoding the extracellular domain may be insertedinto a suitable expression vector for transformation into a prokaryotehost such as E. coli or B. subtilis, to produce a soluble, morphogenbinding fragment. The DNA may expressed directly or may be expressed aspart of a fusion protein having a readily cleavable fusion junction. Anexemplary protocol for prokaryote expression using MR-1 DNA is providedbelow. Recombinant protein is expressed in inclusion bodies and may bepurified therefrom using the technology disclosed in U.S. Pat. No.5,013,653, for example.

The DNA also may be expressed in a suitable mammalian host. Currentlypreferred hosts include fibroblast 3T3 cells, (e.g., NIH 3T3, from CRL1658) COS (simian kidney ATCC, CRL-1650) or CHO (Chinese hamster ovary)cells (e.g., CHO-DXB11, from Lawrence Chasin, Columbia University,N.Y.). An exemplary protocol for mammalian cell expression of MR-1 DNAin 3T3 cells is provided below. Other useful eukaryotic cell systemsinclude yeast cells, the insect/baculovirus system or myeloma cells.

B. MR-1 Expression in E. coli

The extracellular domain of MR-1 was expressed in E. coli. The MR-1 DNAsequence of Seq. ID No.1 was mutagenized in vitro using a "mutagene" kit(BioRad Laboratories, Inc., Richmond) to introduce a methionine (ATG)just before the first serine of the extracellular ligand binding domain(residue 25 in ID No. 1), and to introduce a stop codon after amino acid147.This mutant DNA form was then cloned into a standard commerciallyavailable expression vector, (e.g., pET 3a vector, Novagen, Inc.Wisconsin, opened at NcoI and BamH1), and transfected into a bacterialcell line (e.g., BL21 DE3 pLys S cells, Novagen, Inc.) and expressedusing standard procedures well described in the art. See, for example,Studier et al., (1990) Methods in Enzymology 185:60-89. Proteins thenwere isolated from inclusion bodies using standard procedures: Cellswere grown overnight in the presence of 1 mM IPTG, lysed, and inclusionbodies isolated by centrifugation. Proteins then were resolubilized andpassed over a TSK300 gel filtration column to partially purify theprotein. The protein can be further purified, if desired, by gelelectrophoresis. This form of the protein was used as antigen materialfor antibody production (see below.)

C. MR-1 Expression in CHO Cells

To express the MR-1 protein, the MR-1 DNA is subcloned into an insertionsite of a suitable, commercially available vector (e.g., pCDM8,Invitrogen, Inc. San Diego), along with suitable promoter/enhancersequences and 3' termination sequences. A currently preferredpromoter/enhancer sequence combination includes the CMV promoter (humancytomegalovirus (MIE) promoter) present, for example, on pCDM8, as wellas the mammary tumor virus promoter (mMTV) boosted by the rous sarcomavirus LTR enhancer sequence (e.g., from Clontech, Inc., Palo Alto).Expression also may be further enhanced using transactivating enhancersequences. The plasmid also preferably contains DHFR as an amplifiablemarker, under SV40 early promoter control (ATCC #37148). Transfection,cell culturing, gene amplification and protein expression conditions arestandard conditions, well known in the art, such as are described, forexample in Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989). Briefly, transfected cells are culturedin medium containing 5-10% dialyzed fetal calf serum (FCS), and stablytransfected high expression cell lines obtained by amplificationsubcloning and evaluated by standard Western and Northern blot. Southernblots also can be used to assess the state of integrated receptorsequences and the extent of their copy number amplification.

The expressed protein then is purified using standard procedures. Acurrently preferred methodology uses an affinity column, such as aligand affinity column or an antibody affinity column, the boundmaterial then washed, and receptor molecules selectively eluted in agradient of increasing ionic strength. Alternatively, where a usefulanchor sequence has been added to the DNA, such as a (His)₆ sequence,the column may be a standard affinity column such as Cu²⁺ IMAC column.Here, for example, the cell culture media containing the recombinantprotein is passed over a Cu²⁺ IMAC column prepared with 25 mM imidazol.The bound protein then is washed with a compatible solution and elutedwith EDTA, and the anchor sequence removed by a standard chemical orenzymatic procedure.

Mammalian cell expression is preferred where morphogen receptorexpression on a cell surface is desired. For example, cell surfaceexpression may be desired to test morphogen or morphogen analog bindingspecificity for a cell surface receptor under in vivo conditions. Cellsurface expression also may be most efficacious for high flux screenassays as described below.

EXAMPLE 3 MORPHOGEN RECEPTOR ANTIBODY PRODUCTION

A. General Considerations

Recombinantly produced MR can be used to obtain antibodies capable ofspecifically binding the receptor molecules and useful in immunoassaysand in the immunopurification of morphogen receptors and as describedabove. Using the ligand binding assays described below, antibodies maybe obtained which specifically recognize and interact with the receptorbinding domain, and may be useful as morphogen analogs as described inExample 8, below.

Polyclonal antibodies specific for a morphogen receptor of interest maybe prepared as described below for MR-1. Each rabbit is given a primaryimmunization (e.g., 500 μg) of recombinantly-produced MR-1 protein orprotein fragment (e.g., extracellular domain, "ECD") in 0.1% SDS mixedwith 500 μl Complete Freund's Adjuvant. The antigen is injectedintradermally at multiple sites on the back and flanks of the animal.The rabbit is boosted after a month in the same manner using incompleteFreund's Adjuvant. Test bleeds are taken from the ear vein seven dayslater. Two additional boosts and test bleeds are performed at monthlyintervals until antibody against MR-1 is detected in the serum using astandard Western blot. Then, the rabbit is boosted monthly with 100μg/ml of antigen and bled (15 ml per bleed) at days seven and ten afterboosting.

Similarly, monoclonal antibody specific for a given morphogen receptormolecule of interest may be prepared as described below for MR-1: Amouse is given two injections of MR-1 protein or a protein fragmentspecific for MR-1. The protein preferably is recombinantly produced.Where it is desired that the antibody recognize an epitope on themorphogen binding surface an MR-1 fragment derived from theextracellular domain preferably is provided. The first injectioncontains 100 μg of MR-1 in complete Freund's adjuvant and is givensubcutaneously. The second injection contains 50 μg of MR-1 inincomplete adjuvant and is given intraperitoneally. The mouse thenreceives a total of 230 μg of MR-1 in four intraperitoneal injections atvarious times over an eight month period. One week prior to fusion, themouse is boosted intraperitoneally with MR-1 (e.g., 100 μg) and may beadditionally boosted with an MR-1-specific peptide (e.g., correspondingto a portion of the extracellular domain) conjugated to bovine serumalbumin with a suitable crosslinking agent. This boost can be repeatedfive days (IP), four days (IP), three days (IP) and one day (IV) priorto fusion. The mouse spleen cells then are fused to commerciallyavailable myeloma cells at a ratio of 1:1 using PEG 1500 (BoehringerMannheim, Germany), and the fused cells plated and screened forMR-1-specific antibodies using MR-1 as antigen. The cell fusion andmonoclonal screening steps readily are performed according to standardprocedures well described in standard texts widely available in the art.(See, for example, Guide to Protein Purification Murray P. Deutscher,ed., Academic Press, San Diego, 1990.

B. MR-1 Antisera

MR-1 antiserum was obtained using recombinantly produced soluble MR-1 asantigen and the polyclonal antibody production protocol described above.Soluble MR-1 was expressed from a DNA fragment derived from bases 25 to147 of Seq. ID No.1, as described in Example 2B. Antiserum reactedspecifically with both the E. coli-produced and CHO-produced MR-1proteins as determined by Western blot.

EXAMPLE 4 LIGAND BINDING ASSAYS

Ligand binding specificity is determined by evaluating the ability of areceptor molecule to bind a specific ligand, and the ability of thatligand to compete against itself and other molecules which bind thereceptor. Useful ligands will have a binding affinity for a solublemorphogen receptor extracellular domain such that dissociation constant(Kd) is less than about 10⁻⁷ M, preferably less than 10⁻⁸ M. Relatedmorphogens are expected to be able to bind with specificity to a singlegiven receptor molecule, although likely with differing affinities. Thisability to "crosstalk" is demonstrated by the fact that dpp and 60A, twodrosophila morphogens, both induce bone formation in a rat endochondralbone formation assay, normally induced with OP-1 or BMP2 homodimers ormixtures thereof. dpp is believed to be the drosophila homolog of BMP2,and 60A is believed to be the drosophila homolog of OP-1. In addition,introduction of BMP4 into a dpp- drosophila mutant genome is thought tocompensate for the dpp- mutation in developing flies.

Ligand binding specificity can be assayed as follows, essentiallyfollowing standard protocols well described in the art and disclosed,for example, in Legerski et al. (1992) Biochem. Biophys. Res.Comm183:672-679 and Frakar et al., (1978) Biochem. Biophys. Res.Comm.80:849-857.In the ligand binding assays, a ligand having a known,quantifiable affinity for a morphogen receptor molecule of interest islabelled, typically by radioiodination (¹²⁵ I) or by metaboliclabelling, e.g., ³⁵ S, and aliquots of cells expressing the receptor ontheir surface are incubated with the labelled ligand, in the presence ofvarious concentrations of unlabelled competitor ligand. In the assaysdescribed in Example 8, below, this competitor typically is thecandidate morphogen analog or an aliquot from a broth or extract thatmay contain a candidate morphogen analog.

Briefly, cells expressing MR-1 on their cell surface are plated into 35mM dishes and incubated for 48 hours in DMEM (Dulbecco's modified Eaglemedium) plus 10% fetal calf serum. Purified morphogen, here, e.g.,OP-1,60A or dpp is iodinated with Na¹²⁵ I by chloramin T oxidation,preferably having a specific activity of about 50-100 μCi/μg,essentially following the protocol of Frolik et al. (1984) J. Biol.Chem. 595:10995-11000. Labelled morphogen then is purified usingstandard procedures, e.g., chromatographically. Plated cells are thenwashed twice with physiologically buffered saline in the presence of0.1% BSA, and incubated at 22° C. in the presence of BSA, buffer andlabelled morphogen (1 ng) and various concentrations (e.g., 0-10 μg/ml)of unlabelled competitor, e.g., unlabelled morphogen or candidate ligandanalogs. Following binding, cells are washed three times with coldbuffer, solubilized in 0.5 ml of 0.5N NaOH, removed from the dish, andradioactivity determined by gamma or scintillation counter. Data thenare expressed as percent inhibition, where 100% inhibition of specificbinding is the difference between binding in the absence of competitorand binding in the presence of a 100-fold molar excess of unlabelledmorphogen or candidate ligand. Binding parameters preferably aredetermined using a computer program such as LIGAND (Munsun et al. (1980)Anal. Biochem. 107:220-259.)

Where the receptor cell surface binding domain is to be provided as asoluble protein, the assay may be performed in solution, most readily asan immunoprecipitation assay. In currently preferred assays the receptormolecule is labelled and incubated with unlabelled morphogen orcandidate morphogen analogs. Morphogen-specific antibody then isprovided to the solution to precipitate the receptor-morphogen complexand the amount of labelled morphogen in the precipitated complexdetermined using standard detection means.

In the morphogen analog screening assays described in Example 8 below,the preferred protocol is a standard competition or radioimmunoassay(RIA). Here the morphogen is labelled and the relative binding affinityof a candidate ligand in a sample is measured by quantitating theability of the candidate (unlabelled ligand analog) to inhibit bindingof the labelled ligand (competitor morphogen) by the receptor. Inperforming the assay, fixed concentrations of receptor and labelledmorphogen are incubated in the absence and presence of unknown samplescontaining candidate ligands. Sensitivity may be increased bypreincubating the receptor with candidate ligand before adding thelabelled morphogen. After the labelled competitor has been added,sufficient time is allowed for adequate competitor binding, and thenfree and bound labelled morphogen are separated, and one or the other ismeasured. Useful morphogen labels include radioactive labels andconjugated enzymes having high turnover numbers, such as horseradishperoxidase, alkaline phosphatase, or β-galactosidase, used incombination with chemiluminescent or fluorogenic substrates.

Morphogens useful in binding assays may tested include the matureprotein forms complexed with their pro domains, mature protein formsalone, and truncated forms comprising essentially just the C-terminalactive domain.

EXAMPLE 5 CHIMERIC RECEPTOR MOLECULES

Chimeric MR molecules, e.g., comprising an MR-1 extracellular andtransmembrane region and, for example, part or all of an intracellulardomain from another, different morphogen receptor or an intracellulardomain from a different cell surface molecule, may be constructed usingstandard recombinant DNA technology and/or an automated DNA synthesizerto construct the desired sequence. As will be appreciated by personsskilled in the art, useful junctions include sequences within thetransmembrane region and/or sequences at the junction of either theintracellular or the extracellular domains. Also envisioned are chimerswhere the extracellular domain or the intracellular domains themselvesare chimeric sequences.

Chimeric sequences are envisioned to be particularly useful in screeningassays to determine candidate binding ligands (e.g., morphogen analogs,see below), where the non-MR-1 intracellular domain provides a suitablesecond messenger response system that is easy to detect. Potentiallyuseful other second messenger response systems include those which, whenactivated, induce phosphoinositide hydrolysis, adenylate cyclase,guanylate cyclase or ion channels.

Chimeric receptor molecules have particular utility in gene therapyprotocols. For example, a population of cells expressing a chimericmorphogen receptor molecule their surface and competent for expressing adesired phenotype may be implanted in a mammal at a particular tissuelocus. By careful choice of the ligand binding domain used on thesereceptors a physician can administer to the individual a morphogenagonist capable of binding to the chimeric receptor alone andstimulating the proliferation and/or differentiation of the implantedcells without affecting endogenous cell populations.

EXAMPLE 6 CONSIDERATIONS FOR IDENTIFYING OTHER MORPHOGEN RECEPTORS INNUCLEIC ACID LIBRARIES

Identification of MR-1 allows one to identify other morphogen receptorsequences in different species as well as in different tissues. The MR-1sequence itself can be used as a probe or the sequence may be modifiedto account for a preferred codon bias (e.g., human codon bias.)Currently preferred sequences are those which encode the extracellulardomain.

Probes based on the nucleic acid sequence of Seq. ID No.1 can besynthesized on commercially available DNA synthesizers, e.g. AppliedBiosystems model 381A, using standard techniques, e.g. Gait,Oligonucleotide Synthesis: A Practical Approach, (IRL Press, WashingtonD.C., 1984). It is preferable that the probes are at least 8-50 baseslong, more preferably 18-30 bases long. Probes can be labeled in avariety of ways standard in the art, e.g. using radioactive, enzymaticor colormetric labels as described, for example, by Berent et al,(May/June 1985) Biotechniques: 208-220; and Jablonski et al, (1986)Nucleic Acids Research 14: 6115-6128.

Preferably, low stringency conditions are employed when screening alibrary for morphogen receptor sequences using a probe derived fromMR-1. Preferred MR-1-specific probes are those corresponding to basesencoding the extracellular domain ("ECD"), or encoding a unique(nonhomologous) sequence within the cytoplasmic domain. Useful probesmay be designed from bases 450-820, for example. The probe may befurther modified to use a preferred species codon bias, e.g., a humancodon bias, for example.

For example, for a probe of about 20-40 bases a typicalprehybridization, hybridization, and wash protocol is as follows: (1)prehybridization: incubate nitrocellulose filters containing thedenatured target DNA for 3-4 hours at 55° C. in 5× Denhardt's solution,6× SSC (20× SSC consists of 175 g NaCl, 88.2 g sodium citrate in 800 mlH₂ O adjusted to pH. 7.0 with 10N NaOH), 0.1% SDS, and 100 μg/mldenatured salmon sperm DNA, (2) hybridization: incubate filters inprehybridization solution plus probe at 42° C. for 14-48 hours, (3)wash; three 15 minutes washes in 6× SSC and 0.1% SDS at roomtemperature, followed by a final 1-1.5 minutes wash in 6× SSC and 0.1%SDS at 55° C. Other equivalent procedures, e.g. employing organicsolvents such as formamide, are well known in the art.

Alternatively, morphogen receptor-specific DNA can be amplified using aPCR methodology such as the one disclosed herein, to amplifyapproximately 500 base pair fragments. As for the hybridizationscreening probes described above, the primer sequences preferably arederived from sequences encoding the extracellular domain, or from uniquesequences occurring in the intracellular domain. The primers disclosedherein also are envisioned to be useful.

The invention also provides means for isolating transcriptionalregulatory elements, particularly those occurring 5' and 3' to thecoding sequence, as well as other, nontranslated sequences affectingstability, processing, transcription, translation, and tissuespecificity. For example, using a probe derived from the 5' terminus ofthe sequence of Seq. ID No. 1, upstream untranslated sequences can beobtained. Similarly, using a sequence derived from the 3' terminus,downstream untranslated sequences can be obtained.

EXAMPLE 7 TISSUE DISTRIBUTION OF MORPHOGEN RECEPTORS

Determining the tissue distribution of morphogen receptors may be usedto identify different morphogen receptors expressed in a given tissue,and to identify new, related receptor molecules, as well as to identifytarget tissues for specific morphogens under naturally occurringconditions. The morphogen receptor molecules (or their mRNA transcripts)readily are identified in different tissues using standard methodologiesand minor modifications thereof in tissues where expression may be low.For example, protein distribution may be determined using standardWestern blot analysis or immunodetection techniques, and antibodiesspecific to the morphogen receptor molecules of interest. Similarly, thedistribution of morphogen receptor transcripts may be determined usingstandard Northern hybridization protocols and transcript-specific probesor by in situ hybridization.

Any probe capable of hybridizing specifically to a transcript, anddistinguishing the transcript of interest from other related transcriptsmay be used. Because the morphogen receptors described herein likelyshare high sequence homology in their intracellular domains, the tissuedistribution of a specific morphogen receptor transcript may best bedetermined using a probe specific for the extracellular domain of themolecule. For example, a particularly useful MR-1-specific probesequence is one derived from a portion of the 5' coding sequence. Thechosen fragment then is labelled using standard means well known anddescribed in the art and herein.

Using these morphogen-specific probes, which may be syntheticallyengineered or obtained from cloned sequences, morphogen receptortranscripts can be identified in various tissues of various organisms,using standard methodologies well known to those having ordinary skillin the art. A detailed description of a suitable hybridization protocolis described in Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn.179:116-123, and Ozkaynak, et al. (1992) J Biol. Chem. 267:25220-25227.Briefly, total RNA is prepared from various tissues (e.g., murine embryoand developing and adult liver, kidney, testis, heart, brain, thymus,stomach) by a standard methodologies such as by the method ofChomczynski et al. ((1987) Anal. Biochem162:156-159) and describedbelow. Poly (A)+ RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+ RNA (generally 15 μg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe is denaturedby heating. The hybridization is carried out in a lucite cylinderrotating in a roller bottle apparatus at approximately 1 rev/min forapproximately 15 hours at 37° C. using a hybridization mix of 40%formamide, 5× Denhardts, 5× SSPE, and 0.1% SDS. Following hybridization,the non-specific counts are washed off the filters in 0.1× SSPE, 0.1%SDS at 50° C.

EXAMPLE 8 MORPHOGEN ANALOG SCREENING ASSAYS

The present invention is useful to determine whether a ligand, such as aknown or putative drug, is capable of binding to and/or activating amorphogen cell surface receptor as described herein. Ligands capable ofspecific binding interaction with a given morphogen receptor arereferred to herein as morphogen analogs and may be used for therapeuticand diagnostic applications. Some morphogen analogs will have theability to stimulate morphogenetic activity in the cell, these aremorphogen agonists. Others will have strong binding affinity but willnot stimulate morphogenesis, these are morphogen antagonists. Theseanalogs may be amino acid-based, or they may be composed ofnon-proteinaceous chemical structures.

Transfection of an isolated clone encoding a morphogen receptor into thecell systems described above provides an assay system for the ability ofligands to bind to and/or to activate the receptor encoded by theisolated DNA molecule. Transfection systems, such as those describedabove, are useful as living cell cultures for competitive binding assaysbetween known or candidate drugs and ligands which bind to the receptorand compete with the binding of known morphogens, which are labeled byradioactive, enzymatic, spectroscopic or other reagents. Membranepreparations containing the receptor and isolated from transfected cellsare also useful in these competitive binding assays. Alternatively, andcurrently preferred, purified receptor molecules can be plated onto amicrotiter well surface, in a modification of a sandwich assay, e.g., asa competition assay, such as an RIA, described above. Finally, asdescribed above, solution assays, and using only the receptorextracellular domain, also may be used to advantage in these assays.Functional assays of second messenger systems or their sequelae intransfection systems act as assays for binding affinity and efficacy inthe activation of receptor function. Such a transfection systemconstitutes a "drug discovery system", useful for the identification ofnatural or synthetic compounds with potential for drug development thatcan be further modified or used directly as therapeutic compounds toactivate or inhibit the natural functions of the receptor encoded by theisolated DNA molecule.

Two approaches to identifying morphogen receptor binding analogstypically are practiced in the art: high flux screens and rationaldesign of ligand mimetics. High flux screens typically screen naturallysourced materials or chemical banks for their ability to bind a proteinof interest, here, the receptor. Typically, compounds are obtained froma range of sources, e.g., chemical banks, microbial broths, plant andanimal extracts, and the like. In a high flux screen typically, purifiedreceptor is plated onto a microtiter well surface and a standard volumeof a sample solution to be tested then is added. Also added is astandard volume having a known quantity of a purified ligand known tobind the receptor with specificity. Preferably the ligand is labelledwith a substance that is readily detectable by automated means (e.g.,radiolabel, chromophoric, enzymatic or spectroscopic label.) The wellsthen are washed and the amount of label remaining after washing or theamount of label remaining associated with the receptor then is detected.Positive scores are identified by the ability of the test substance toprevent interaction of the labelled ligand with the receptor. Where MR-1is the receptor, useful ligands for labelling include OP-1, 60A and DPP.High flux screens exploit both the high degree of specificity of thelabelled ligand for its receptor, as well as high throughput capacity ofcomputer driven robotics and computer handling of data. Candidateanalogs identified in this manner, then can be analyzed structurally andthis information used to design and to synthesize analogs havingenhanced potency, increased duration of action, increased selectivityand reduced side effects. Candidates also can be used in a rationaldesign program as described below. Finally, candidate analogs also canbe tested to determine morphogenetic effect, if any, as described below.

The second approach to the identification of morphogen analogs uses arational design approach to create molecules capable of mimicking thebinding effect of morphogens with their receptors. Here the relevantstructure for receptor binding is analyzed to identify criticalsequences and structures necessary for binding activity and thisinformation can be used to design and synthesize minimal size morphogenanalogs. As for candidate compounds in the high flux assay, designcandidates can be tested for ligand activity as described above.

Antibodies capable of interacting specifically with the morphogenreceptor and competing with morphogen binding also may be used asmorphogen analogs. Antibodies may be generated as described above.

Morphogen analogs may be evaluated for their ability to mimic morphogensor to inhibit morphogens (e.g., morphogen agonists or antagonists) bymonitoring the effect of the analogs on cells bearing the appropriatemorphogen receptors. Morphogen agonists are anticipated to have utilityin any application where tissue morphogenesis is desired, such as in theregeneration of damaged tissue resulting from mechanical or chemicaltrauma, degenerative diseases, tissue destruction resulting from chronicinflammation, cirrhosis, inflammatory diseases, cancer and the like, andin the regeneration of tissues, organs and limbs. Morphogen antagonistsare envisioned to have utility in applications where tissuemorphogenesis is to be limited as, for example, in the treatment ofmalignant transformations including, but not limited to, osteosarcomasand Paget's disease.

For example, morphogens are known to preferentially inducedifferentiation of progenitor cells, including embryonic mesenchymalcells and primary osteoblasts (see, for example, PCT US92/07432 (nowpublished as WO 93/05751), the disclosure of which is incorporatedherein by reference.) As one example, morphogen analogs can be testedfor their ability to induce differentiation of primary osteoblasts, bymeasuring the ability of these analogs to induce production of alkalinephosphatase, PTH-mediated cAMP and osteocalcin, all of which are inducedwhen primary osteoblasts are exposed to OP-1, 60A or DPP.

Briefly, the assays may be performed as follows. In this and allexamples involving osteoblast cultures, rat osteoblast-enriched primarycultures preferably are used. Although these cultures are heterogeneousin that the individual cells are at different stages of differentiation,the culture is believed to more accurately reflect the metabolism andfunction of osteoblasts in vivo than osteoblast cultures obtained fromestablished cell lines. Unless otherwise indicated, all chemicalsreferenced are standard, commercially available reagents, readilyavailable from a number of sources, including Sigma Chemical, Co., St.Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co., Milwaukee.

Rat osteoblast-enriched primary cultures are prepared by sequentialcollagenase digestion of newborn suture-free rat calvaria (e.g., from1-2 day-old animals, Long-Evans strain, Charles River Laboratories,Wilmington, Mass.), following standard procedures, such as aredescribed, for example, in Wong et al., (1975) PNAS72:3167-3171. Ratosteoblast single cell suspensions then are plated onto a multi-wellplate (e.g., a 24 well plate) at a concentration of 50,000 osteoblastsper well in alpha MEM (modified Eagle's medium, Gibco, Inc., LongIsland) containing 10% FBS (fetal bovine serum), L-glutamine andpenicillin/streptomycin. The cells are incubated for 24 hours at 37° C.,at which time the growth medium is replaced with alpha MEM containing 1%FBS and the cells incubated for an additional 24 hours so that the cellsare in serum-deprived growth medium at the time of the experiment.

Alkaline Phosphatase Induction of Osteoblasts

The cultured cells in serum-free medium are incubated with morphogen(e.g., OP-1), morphogen analog or a negative control, using a range ofconcentrations. For example, 0.1, 1.0, 10.0, 40.0 or 80.0 ng OP-1/mlmedium typically are used. 72 hours after the incubation period the celllayer is extracted with 0.5 ml of 1% Triton X-100.The resultant cellextract then, is centrifuged, and 100 μl of the extract is added to 90μl of paranitrosophenylphospate (PNPP)/glycerine mixture and incubatedfor 30 minutes in a 37° C. water bath and the reaction stopped with 100μl NaOH. The samples then are run through a plate reader (e.g., DynatechMR700 plate reader, and absorbance measured at 400 nm, usingp-nitrophenol as a standard) to determine the presence and amount ofalkaline phosphate activity. Protein concentrations are determined bythe Biorad method. Alkaline phosphatase activity is calculated inunits/μg protein, where 1 unit=1 nmol p-nitrophenol liberated/30 minutesat 37° C. OP-1 induces a five-fold increase in the specific activity ofalkaline phosphate by this method. Agonists are expected to have similarinduction effects. Antagonists should inhibit or otherwise interferewith morphogen binding, and diminished alkaline phosphatase inductionshould result when the assay is performed with an antagonist in thepresence of a limiting amount of morphogen.

Induction of PTH-Mediated cAMP.

The effect of a morphogen analog on parathyroid hormone-mediated cAMPproduction in rat osteoblasts in vitro may be demonstrated as follows.

Rat osteoblasts are prepared and cultured in a multiwell plate asdescribed above. The cultured cells then are divided into three groups:(1) wells which receive, for example, 1.0, 10.0 and 40.0 ng OP-1/mlmedium); (2) wells which receive the candidate analog at variousconcentration ranges; and (3) a control group which receives noadditional factors. The plate is then incubated for another 72 hours. Atthe end of the 72 hours the cells are treated with medium containing0.5% bovine serum albumin (BSA) and 1 mM 3-isobutyl-1-methylxanthine for20 minutes followed by the addition into half of the wells of humanrecombinant parathyroid hormone (hPTH, Sigma, St. Louis) at aconcentration of 200 ng/ml for 10 minutes. The cell layer then isextracted from each well with 0.5 ml of 1% Triton X-100. The cAMP levelsthen are determined using a radioimmunoassay kit (e.g., Amersham,Arlington Heights, Ill.). OP-1 doubles cAMP production in the presenceof PTH. Agonists are expected to have similar induction effects.Antagonists are expected to inhibit or otherwise interfere withmorphogen binding, and diminished cAMP production should result when theassay is performed with an antagonist in the presence of limiting theamount of morphogen.

Induction of Osteocalcin Production

Osteocalcin is a bone-specific protein synthesized by osteoblasts whichplays an integral role in the rate of bone mineralization in vivo.Circulating levels of osteocalcin in serum are used as a marker forosteoblast activity and bone formation in vivo. Induction of osteocalcinsynthesis in osteoblast-enriched cultures can be used to demonstratemorphogenic efficacy in vitro.

Rat osteoblasts are prepared and cultured in a multi-well plate asabove. In this experiment the medium is supplemented with 10% FBS, andon day 2, cells are fed with fresh medium supplemented with fresh 10 mMβ-glycerophosphate (Sigma, Inc.). Beginning on day 5 and twice weeklythereafter, cells are fed with a complete mineralization mediumcontaining all of the above components plus fresh L(+)-ascorbate, at afinal concentration of 50 μg/ml medium. Morphogen or morphogen analogthen is added to the wells directly, e.g., in 50% acetonitrile (or 50%ethanol) containing 0.1% trifluoroacetic acid (TFA), at no more than 5μl morphogen/ml medium. Control wells receive solvent vehicle only. Thecells then are re-fed and the conditioned medium sample diluted 1:1 instandard radioimmunoassay buffer containing standard protease inhibitorsand stored at -20° C. until assayed for osteocalcin. Osteocalcinsynthesis is measured by standard radioimmunoassay using a commerciallyavailable osteocalcin-specific antibody and can be confirmed by Northernblot analysis to calculate the amount of osteocalcin mRNA produced inthe presence and absence of OP-1 or morphogen analog. OP-1 induces adose-dependent increase in osteocalcin production (5-fold increase using25 ng of OP-1 protein/ml), and a 20-fold increase in osteocalcin mRNA.Agonists are expected to have similar induction effects; antagonists areexpected to inhibit or otherwise interfere with morphogen binding,thereby substantially interfering with osteocalcin induction in thepresence of a limiting amount of morphogen.

Mineralization is determined on long term cultures (13 day) using amodified von Kossa staining technique on fixed cell layers: cells arefixed in fresh 4% paraformaldehyde at 23° C. for 10 min, followingrinsing cold 0.9% NaCl. Fixed cells then are stained for endogenousalkaline phosphatase at pH 9.5 for 10 min, using a commerciallyavailable kit (Sigma, Inc.) Purple stained cells then are dehydratedwith methanol and air dried. After 30 min incubation in 3% AgNO₃ in thedark, H₂ O-rinsed samples are exposed for 30 sec to 254 nm UV light todevelop the black silver-stained phosphate nodules. Individualmineralized foci (at least 20 μm in size) are counted under a dissectingmicroscope and expressed as nodules/culture. OP-1 induces a 20-foldincrease in initial mineralization rate. Agonists are expected to havesimilar induction effects; antagonists are expected to inhibit orotherwise interfere with morphogen binding, thereby inhibitingmineralization induction in the presence of a limiting amount ofmorphogen.

EXAMPLE 9 SCREENING ASSAY FOR COMPOUNDS WHICH ALTER ENDOGENOUS MORPHOGENRECEPTOR LEVELS

Candidate compound(s) which may be administered to affect the level of agiven endogenous morphogen receptor may be found using the followingscreening assay, in which the level of morphogen receptor production bya cell type which produces measurable levels of the receptor isdetermined by incubating the cell in culture with and without thecandidate compound, in order to assess the effects of the compound onthe cell. This can be accomplished by detection of the morphogenreceptor either at the protein or RNA level. The protocol is based on aprocedure for identifying compounds which alter endogenous levels ofmorphogen expression, a detailed description also may be found in (PCTUS92/07359 published as WO 93/05172), incorporated herein by reference.

Cell cultures of kidney, adrenals, urinary bladder, brain, or otherorgans, may be prepared as described widely in the literature. Forexample, kidneys may be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues may be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells may be cultured, for example, in Dulbecco's Modified Eaglemedium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calfserum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or indefined medium (e.g., containing insulin, transferrin, glucose, albumin,or other growth factors).

Cell samples for testing the level of morphogen receptor production arecollected periodically and evaluated for receptor production byimmunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), or, alternatively,a portion of the cell culture itself can be collected periodically andused to prepare polyA+ RNA for RNA analysis. To monitor de novo receptorsynthesis, some cultures are labeled according to conventionalprocedures with an ³⁵ S-methionine/³⁵ S-cysteine mixture for 6-24 hoursand then evaluated to quantitate receptor synthesis by conventionalimmunoassay methods. Alternatively, anti-receptor antibodies may belabelled and incubated with the cells or cell lysates, and the boundcomplexes detected and quantitated by conventional means, such as thosedescribed hereinabove. Northern blots may be performed using a portionof the morphogen receptor coding sequence to create hybridizationprobes, and following the RNA hybridization protocol described herein.

EXAMPLE 10 GENERAL MORPHOGEN ANALOG FORMULATION/ADMINISTRATIONCONSIDERATIONS

The morphogen analogs identified using the methodology described hereinmay be provided to an individual by any suitable means, preferablydirectly or systemically, e.g., parenterally or orally. Where themorphogen analog is to be provided directly (e.g., locally, as byinjection, to a desired tissue site), or parenterally, such as byintravenous, subcutaneous, intramuscular, intraorbital, ophthalmic,intraventricular, intracranial, intracapsular, intraspinal,intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal orby aerosol administration, the analog preferably comprises part of anaqueous solution. The solution preferably is physiologically acceptableso that in addition to delivery of the desired morphogen to the patient,the solution does not otherwise adversely affect the patient'selectrolyte and volume balance. The aqueous medium for the morphogenanalog thus may comprise normal physiologic saline (0.9% NaCl, 0.15M),pH 7-7.4 or other pharmaceutically acceptable salts thereof.

Useful solutions for oral or parenteral administration may be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990.Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the morphogen in vivo.

Other potentially useful parenteral delivery systems for these morphogenanalogs include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration may contain as excipients, for example,lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oilysolutions for administration in the form of nasal drops, or as a gel tobe applied intranasally.

Alternatively, the morphogens described herein may be administeredorally.

The morphogen analogs also may be associated with means for targetingthe analog to a desired tissue. For example, tetracycline anddiphosphonates (bisphosphonates) are known to bind to bone mineral,particularly at zones of bone remodeling, when they are providedsystemically in a mammal. Accordingly, these molecules may be includedas useful agents for targeting analogs to bone tissue. Alternatively, anantibody or other binding protein that interacts specifically with asurface molecule on the desired target tissue cells also may be used.Such targeting molecules further may be covalently associated to themorphogen analog e.g., by chemical crosslinking, or by using standardgenetic engineering means to create, for example, an acid labile bondsuch as an Asp-Pro linkage. Useful targeting molecules may be designed,for example, using the single chain binding site technology disclosed,for example, in U.S. Pat. No. 5,091,513.

Finally, morphogen analogs may be administered alone or in combinationwith other molecules known to have a beneficial effect on tissuemorphogenesis, including molecules capable of tissue repair andregeneration and/or inhibiting inflammation. Examples of usefulcofactors for stimulating bone tissue growth in osteoporoticindividuals, for example, include but are not limited to, vitamin D₃,calcitonin, prostaglandins, parathyroid hormone, dexamethasone, estrogenand IGF-I or IGF-II. Useful cofactors for nerve tissue repair andregeneration may include nerve growth factors. Other useful cofactorsinclude symptom-alleviating cofactors, including antiseptics,antibiotics, antiviral and antifungal agents and analgesics andanesthetics.

Morphogen analogs further can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. As noted above, such compositions may beprepared for parenteral administration, particularly in the form ofliquid solutions or suspensions; for oral administration, particularlyin the form of tablets or capsules; or intranasally, particularly in theform of powders, nasal drops or aerosols. Where adhesion to a tissuesurface is desired the composition may include the morphogen dispersedin a fibrinogen-thrombin composition or other bioadhesive such as isdisclosed, for example in PCT US91/09275 (published as WO 92/10567), thedisclosure of which is incorporated herein by reference. The compositionthen may be painted, sprayed or otherwise applied to the desired tissuesurface.

The compositions can be formulated for parenteral or oral administrationto humans or other mammals in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the analog to targettissue for a time sufficient to induce the desired effect.

Where the analog is to be used as part of a transplant procedure, it maybe provided to the living tissue or organ to be transplanted prior toremoval of tissue or organ from the donor. The analog may be provided tothe donor host directly, as by injection of a formulation comprising theanalog into the tissue, or indirectly, e.g., by oral or parenteraladministration, using any of the means described above.

Alternatively or, in addition, once removed from the donor, the organ orliving tissue may be placed in a preservation solution containing themorphogen analog. In addition, the recipient also preferably is providedwith the analog just prior to, or concomitant with, transplantation. Inall cases, the analog may be administered directly to the tissue atrisk, as by injection to the tissue, or it may be provided systemically,either by oral or parenteral administration, using any of the methodsand formulations described herein and/or known in the art.

Where the morphogen analog comprises part of a tissue or organpreservation solution, any commercially available preservation solutionmay be used to advantage. For example, useful solutions known in the artinclude Collins solution, Wisconsin solution, Belzer solution,Eurocollins solution and lactated Ringer's solution. Generally, an organpreservation solution usually possesses one or more of the followingproperties: (a) an osmotic pressure substantially equal to that of theinside of a mammalian cell,(solutions typically are hyperosmolar andhave K+ and/or Mg++ ions present in an amount sufficient to produce anosmotic pressure slightly higher than the inside of a mammalian cell);(b) the solution typically is capable of maintaining substantiallynormal ATP levels in the cells; and (c) the solution usually allowsoptimum maintenance of glucose metabolism in the cells. Organpreservation solutions also may contain anticoagulants, energy sourcessuch as glucose, fructose and other sugars, metabolites, heavy metalchelators, glycerol and other materials of high viscosity to enhancesurvival at low temperatures, free oxygen radical inhibiting and/orscavenging agents and a pH indicator. A detailed description ofpreservation solutions and useful components may be found, for example,in U.S. Pat. No. 5,002,965, the disclosure of which is incorporatedherein by reference.

As will be appreciated by those skilled in the art, the concentration ofthe compounds described in a therapeutic composition will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. The preferreddosage of drug to be administered also is likely to depend on suchvariables as the type and extent of tissue loss or defect, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, the formulation of the compound, thepresence and types of excipients in the formulation, and the route ofadministration. In general terms, the morphogen analogs of thisinvention may be provided to and individual where typical dose rangesare from about 10 ng/kg to about 1 g/kg of body weight per day; apreferred dose range being from about 0.1 μg/kg to 100 mg/kg of bodyweight. No obvious morphogen-induced pathological lesions are inducedwhen mature morphogen (e.g., OP-1, 20 μg) is administered daily tonormal growing rats for 21 consecutive days. Moreover, 10 μg systemicinjections of morphogen (e.g., OP-1) injected daily for 10 days intonormal newborn mice does not produce any gross abnormalities.

Finally, morphogen receptor ligand binding antagonists may be identifiedas described herein having utility as insecticidal agents. Specifically,MR-1 may be used as the receptor to identify receptor binding ligand asdescribed herein antagonists capable of interfering with normal insectdevelopment. Useful morphogen antagonists may be combined with one ormore excipients useful in insecticide formulations. Soluble forms of aninsect-specific morphogen receptor may be useful. Those skilled in theart will appreciate that binding specificity is critical. Preferredbinding affinities have a Kd less than 10⁻⁹ M, preferably less than10⁻¹⁰ M.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2625 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 379..1929                                                       (D) OTHER INFORMATION: /product="DROSOPHILA MORPHOGEN                         RECEPTOR"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CACGAGAAATCGCTTGAAAACAGGCCCGCAGACCTGCGAAAAACGAAAAAGTGCAGCGCG60                CATATACTTTTTCAACTGTGCCCCTCTAGCTTAAAATTAAGTCGCGGCGAAAAGTCGAGT120               AAAAACCGCGGAAATGCGCATGCAAACGGTGTGTGGCCAGCAAAATCGCTGCCAAGGCAC180               CGCACACACACTCGGCCACCCACACATACACACTTAGTGCTGTACTCGAAAAGTGCGAAG240               ACAACAGGAACTCTGTGCCAAAATAATATTAACAGTACCCAGTTATTCCATTCCACTGCA300               CCTGTCCCCGAAACATCGAAATATTCGCGTTACGTATACGCAACGAGTGCTGTAAACAAG360               TTTGCACAGGCGATAACAATGTCCAAATACGATCTGCTTTATCTAACGGCG411                        MetSerLysTyrAspLeuLeuTyrLeuThrAla                                             1510                                                                          CACGTAACGCTGGTCTGCTGTCTGATTGGAATCCATGGATCTATTTTG459                           HisValThrLeuValCysCysLeuIleGlyIleHisGlySerIleLeu                              152025                                                                        CCCGGAAGTCATGGGATCATAGAATGCGAGCACTTCGACGAGAAGATG507                           ProGlySerHisGlyIleIleGluCysGluHisPheAspGluLysMet                              303540                                                                        TGCAACACAACGCAGCAATGTGAAACACGGATAGAGCACTGTAAGATG555                           CysAsnThrThrGlnGlnCysGluThrArgIleGluHisCysLysMet                              455055                                                                        GAGGCGGATAAGTTTCCCAGCTGCTATGTCCTTTGGTCGGTCAACGAG603                           GluAlaAspLysPheProSerCysTyrValLeuTrpSerValAsnGlu                              60657075                                                                      ACAACGGGCATCCTGCGCATCAAGATGAAGGGCTGCTTCACGGACATG651                           ThrThrGlyIleLeuArgIleLysMetLysGlyCysPheThrAspMet                              808590                                                                        CACGAATGCAATCAGACGGAGTGCGTGACCAGTGCAGAGCCACGGCAG699                           HisGluCysAsnGlnThrGluCysValThrSerAlaGluProArgGln                              95100105                                                                      GGAAACATTCACTTCTGCTGCTGCAAGGGATCGCGGTGCAATTCCAAC747                           GlyAsnIleHisPheCysCysCysLysGlySerArgCysAsnSerAsn                              110115120                                                                     CAGAAATATATTAAAAGCACCACGGAGGCAACCACACAAGTGCCCAAG795                           GlnLysTyrIleLysSerThrThrGluAlaThrThrGlnValProLys                              125130135                                                                     GAGAAGACGCAGGACGGCAGCAATTTGATATACATCTACATTGGCACC843                           GluLysThrGlnAspGlySerAsnLeuIleTyrIleTyrIleGlyThr                              140145150155                                                                  TCCGTTTTCAGCGTGCTCATGGTCATTGTTGGCATGGGCCTTCTTCTC891                           SerValPheSerValLeuMetValIleValGlyMetGlyLeuLeuLeu                              160165170                                                                     TACCGACGCCGCAAGCAGGCGCACTTTAACGAGATACCCACGCACGAG939                           TyrArgArgArgLysGlnAlaHisPheAsnGluIleProThrHisGlu                              175180185                                                                     GCTGAGATAACAAACTCATCGCCATTGCTCAGCAACCGTCCCATTCAG987                           AlaGluIleThrAsnSerSerProLeuLeuSerAsnArgProIleGln                              190195200                                                                     CTGCTGGAACAGAAGGCCAGTGGTAGATTCGGTGATGTGTGGCAAGCC1035                          LeuLeuGluGlnLysAlaSerGlyArgPheGlyAspValTrpGlnAla                              205210215                                                                     AAGCTCAACAATCAGGATGTGGCCGTCAAGATCTTTCGCATGCAGGAA1083                          LysLeuAsnAsnGlnAspValAlaValLysIlePheArgMetGlnGlu                              220225230235                                                                  AAAGAATCGTGGACCACGGAGCACGATATCTACAAGCTGCCGCGCATG1131                          LysGluSerTrpThrThrGluHisAspIleTyrLysLeuProArgMet                              240245250                                                                     CGCCATCCGAACATCCTCGAATTCCTGGGCGTTGAGAAGCACATGGAC1179                          ArgHisProAsnIleLeuGluPheLeuGlyValGluLysHisMetAsp                              255260265                                                                     AAGCCGGAATATTGGCTGATATCCACCTACCAGCATAACGGATCACTA1227                          LysProGluTyrTrpLeuIleSerThrTyrGlnHisAsnGlySerLeu                              270275280                                                                     TGCGACTACCTCAAATCGCACACGATCTCATGGCCAGAGTTGTGCCGC1275                          CysAspTyrLeuLysSerHisThrIleSerTrpProGluLeuCysArg                              285290295                                                                     ATCGCTGAGTCCATGGCCAATGGACTGGCACATCTGCACGAGGAGATC1323                          IleAlaGluSerMetAlaAsnGlyLeuAlaHisLeuHisGluGluIle                              300305310315                                                                  CCGGCATCAAAGACCGATGGGCTAAAACCATCGATAGCTCACCGAGAC1371                          ProAlaSerLysThrAspGlyLeuLysProSerIleAlaHisArgAsp                              320325330                                                                     TTCAAGTCTAAGAACGTACTGCTTAAGAGCGATCTGACGGCCTGTATA1419                          PheLysSerLysAsnValLeuLeuLysSerAspLeuThrAlaCysIle                              335340345                                                                     GCTGACTTTGGTTTGGCCATGATATTCCAGCCAGGCAAGCCCTGCGGC1467                          AlaAspPheGlyLeuAlaMetIlePheGlnProGlyLysProCysGly                              350355360                                                                     GATACACACGGTCAAGTAGGCACTCGACGTTACATGGCCCCAGAGGTG1515                          AspThrHisGlyGlnValGlyThrArgArgTyrMetAlaProGluVal                              365370375                                                                     CTTGAGGGTGCCATCAATTTCAATAGAGACGCTTTCTTACGCATAGAC1563                          LeuGluGlyAlaIleAsnPheAsnArgAspAlaPheLeuArgIleAsp                              380385390395                                                                  GTCTACGCATGCGGCCTAGTCCTCTGGGAAATGGTGTCACGGTGTGAC1611                          ValTyrAlaCysGlyLeuValLeuTrpGluMetValSerArgCysAsp                              400405410                                                                     TTTGCCGGACCCGTCGGTGAGTTCCAGCTGCCTTTTGAGGCCGAGCTG1659                          PheAlaGlyProValGlyGluPheGlnLeuProPheGluAlaGluLeu                              415420425                                                                     GGCCTGAGGCCGTCGCTGGACGAAGTTCAGGAGAGTGTGGTAATGAAG1707                          GlyLeuArgProSerLeuAspGluValGlnGluSerValValMetLys                              430435440                                                                     AAGCTGCGCCCTCGTTTGCTCAACTCCTGGCGCGCCCATCCGGGACTT1755                          LysLeuArgProArgLeuLeuAsnSerTrpArgAlaHisProGlyLeu                              445450455                                                                     AATGTATTCTGCGACACAATGGAGGAGTGCTGGGATCACGACGCTGAG1803                          AsnValPheCysAspThrMetGluGluCysTrpAspHisAspAlaGlu                              460465470475                                                                  GCTCGTCTTAGCTCTTCGTGTGTAATGGAACGCTTTGCGCAGCTAAAC1851                          AlaArgLeuSerSerSerCysValMetGluArgPheAlaGlnLeuAsn                              480485490                                                                     AAGTATCCCTCAACCCAGTTGCTGATCAAAAACCACACCAACATTGAC1899                          LysTyrProSerThrGlnLeuLeuIleLysAsnHisThrAsnIleAsp                              495500505                                                                     GACGCCAAGGAATCTACGAATTGCTTATAGAAGCGGTACTAAGCCAC1946                           AspAlaLysGluSerThrAsnCysLeu                                                   510515                                                                        AGACCAGCCAGCGGATCTGTGGCTCTGCAAAAAAATCAGTTGAAGCTTTTTGCTTCGTAG2006              TGGATAGTTGATCGTCGAGGTCGTAAGACGTTTCGTTTGTAGTTTAATAGTTTGTAGTAC2066              CATTATAAGCAGCTACAGAATGGGTTGATTTATACGAAAGAAGTCGTGTAAGGTGTAACT2126              TTTCCTAAGGGAAAAGCTAAAAGCTTAACATGCCAACGATTTTTTGTTATTATTTTCTTT2186              AAATTATAAATCGACTCATTCGATGTTAACCCGAGATCTTAAATCTGTTGGAAATAAGTG2246              GAAACGTACACTAAGCAACTTTGAACAAAGAGCATTAGCTATGTCCAGAGACGTGGTAGT2306              TGCTAATCCGACATTGACTTCATTGACTGATAACCATATATAGTTCTATATAAATCATGC2366              ATATAACGTACAATTTGTTTTGCCCTCTGGCAGTAGTCTGTGTGCATATGTAAACTGTTG2426              AAAATTATCGTCAAGCCTTTTAAAGTATAATTTTATTTGTACGTTATCGAATGTCTTCAT2486              ATAGTAGTAGTTTTATATTTAAAGTTATTTTCAAAATAGATTTAACAATTTTAGTTTACT2546              TTTTAGTTGTAACGTTTTTATTTCGCGCTCAAACGTGCATGCGTTTTAATGTTGTATTTT2606              AAATAAAAAATACTTTAAA2625                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 516 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerLysTyrAspLeuLeuTyrLeuThrAlaHisValThrLeuVal                              151015                                                                        CysCysLeuIleGlyIleHisGlySerIleLeuProGlySerHisGly                              202530                                                                        IleIleGluCysGluHisPheAspGluLysMetCysAsnThrThrGln                              354045                                                                        GlnCysGluThrArgIleGluHisCysLysMetGluAlaAspLysPhe                              505560                                                                        ProSerCysTyrValLeuTrpSerValAsnGluThrThrGlyIleLeu                              65707580                                                                      ArgIleLysMetLysGlyCysPheThrAspMetHisGluCysAsnGln                              859095                                                                        ThrGluCysValThrSerAlaGluProArgGlnGlyAsnIleHisPhe                              100105110                                                                     CysCysCysLysGlySerArgCysAsnSerAsnGlnLysTyrIleLys                              115120125                                                                     SerThrThrGluAlaThrThrGlnValProLysGluLysThrGlnAsp                              130135140                                                                     GlySerAsnLeuIleTyrIleTyrIleGlyThrSerValPheSerVal                              145150155160                                                                  LeuMetValIleValGlyMetGlyLeuLeuLeuTyrArgArgArgLys                              165170175                                                                     GlnAlaHisPheAsnGluIleProThrHisGluAlaGluIleThrAsn                              180185190                                                                     SerSerProLeuLeuSerAsnArgProIleGlnLeuLeuGluGlnLys                              195200205                                                                     AlaSerGlyArgPheGlyAspValTrpGlnAlaLysLeuAsnAsnGln                              210215220                                                                     AspValAlaValLysIlePheArgMetGlnGluLysGluSerTrpThr                              225230235240                                                                  ThrGluHisAspIleTyrLysLeuProArgMetArgHisProAsnIle                              245250255                                                                     LeuGluPheLeuGlyValGluLysHisMetAspLysProGluTyrTrp                              260265270                                                                     LeuIleSerThrTyrGlnHisAsnGlySerLeuCysAspTyrLeuLys                              275280285                                                                     SerHisThrIleSerTrpProGluLeuCysArgIleAlaGluSerMet                              290295300                                                                     AlaAsnGlyLeuAlaHisLeuHisGluGluIleProAlaSerLysThr                              305310315320                                                                  AspGlyLeuLysProSerIleAlaHisArgAspPheLysSerLysAsn                              325330335                                                                     ValLeuLeuLysSerAspLeuThrAlaCysIleAlaAspPheGlyLeu                              340345350                                                                     AlaMetIlePheGlnProGlyLysProCysGlyAspThrHisGlyGln                              355360365                                                                     ValGlyThrArgArgTyrMetAlaProGluValLeuGluGlyAlaIle                              370375380                                                                     AsnPheAsnArgAspAlaPheLeuArgIleAspValTyrAlaCysGly                              385390395400                                                                  LeuValLeuTrpGluMetValSerArgCysAspPheAlaGlyProVal                              405410415                                                                     GlyGluPheGlnLeuProPheGluAlaGluLeuGlyLeuArgProSer                              420425430                                                                     LeuAspGluValGlnGluSerValValMetLysLysLeuArgProArg                              435440445                                                                     LeuLeuAsnSerTrpArgAlaHisProGlyLeuAsnValPheCysAsp                              450455460                                                                     ThrMetGluGluCysTrpAspHisAspAlaGluAlaArgLeuSerSer                              465470475480                                                                  SerCysValMetGluArgPheAlaGlnLeuAsnLysTyrProSerThr                              485490495                                                                     GlnLeuLeuIleLysAsnHisThrAsnIleAspAspAlaLysGluSer                              500505510                                                                     ThrAsnCysLeu                                                                  515                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..194                                                          (D) OTHER INFORMATION: /product="DROSOPHILA GENOMIC DNA                       PROBE USED TO CLONE RECEPTOR CDNA"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATCTGTGGCAGTGAAAATCTTCCCGCATGCAGGAAAAAGAATCGTGGACCACGGAGCA60                CGATATCTACAAGCTGCCGCGCATGGCCATCCGAACATCCTCGAATTCCTGGGCGTTGAG120               AAGCACATGGACAAGCCGGAATATTGGCTGATATCCACCTACCAGCATAACGGTATCACT180               ATGCGTCTTCCTAG194                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: /product="PRIMER 1"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATTSKWRCTYTTGADGTCSCKGTG24                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: /product="PRIMER 2"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATYGCBCACMGSGAYHTCAARAG23                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: /product="PRIMER 3"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAATCTGTSGCHGTSAARRTHTTYCC26                                                  (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: /product="PRIMER 4"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCCAGSACYTCNGGDGCCAKRTA23                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 669 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..669                                                          (D) OTHER INFORMATION: /note= "C ELEGANS RECEPTOR KINASE"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetArgIleArgHisValValPheCysLeuLeuAlaLeuValTyrGly                              151015                                                                        AlaGluThrSerAspAspAspLeuAspGluArgThrAsnIlePheIle                              202530                                                                        ArgAspLysLeuIleProAlaLeuLysLeuAlaGluValThrLysVal                              354045                                                                        AsnPheThrArgLeuHisLeuCysHisCysSerArgGluValGlyCys                              505560                                                                        AsnAlaArgThrThrGlyTrpValProGlyIleGluPheLeuAsnGlu                              65707580                                                                      ThrAspArgSerPheTyrGluAsnThrCysTyrThrAspGlySerCys                              859095                                                                        TyrGlnSerAlaArgProSerProGluIleSerHisPheGlyCysMet                              100105110                                                                     AspGluLysSerValThrAspGluThrGluPheHisAspThrAlaAla                              115120125                                                                     LysValCysThrAsnAsnThrLysAspProHisAlaThrValTrpIle                              130135140                                                                     CysCysAspLysGlyAsnPheCysAlaAsnGluThrIleIleHisLeu                              145150155160                                                                  AlaProGlyProGlnGlnSerSerThrTrpLeuIleLeuThrIleLeu                              165170175                                                                     AlaLeuLeuThrPheIleValLeuLeuGlyIleAlaIlePheLeuThr                              180185190                                                                     ArgLysSerTrpGluAlaLysPheAspTrpTyrIleArgPheLysPro                              195200205                                                                     LysProGlyAspProLeuArgGluThrGluAsnAsnValProMetVal                              210215220                                                                     ThrMetGlyAspGlyAlaGlySerSerValProGluValAlaProIle                              225230235240                                                                  GluGlnGlnGlySerThrMetSerThrSerAlaGlyAsnSerPhePro                              245250255                                                                     ProGlyIleMetProAsnAsnMetLysAspMetLeuAspValLeuGlu                              260265270                                                                     GluThrSerGlySerGlyMetGlyProThrThrLeuHisLysLeuThr                              275280285                                                                     IleGlyGlyGlnIleArgLeuThrGlyArgValGlySerGlyArgPhe                              290295300                                                                     GlyAsnValSerArgGlyAspTyrArgGlyGluAlaValAlaValLys                              305310315320                                                                  ValPheAsnAlaLeuAspGluProAlaPheHisLysGluThrGluIle                              325330335                                                                     PheGluThrArgMetLeuArgHisProAsnValLeuArgTyrIleGly                              340345350                                                                     SerAspArgValAspThrGlyPheValThrGluLeuTrpLeuValThr                              355360365                                                                     GluTyrHisProSerGlySerLeuHisAspPheLeuLeuGluAsnThr                              370375380                                                                     ValAsnIleGluThrTyrTyrAsnLeuMetArgSerThrAlaSerGly                              385390395400                                                                  LeuAlaPheLeuHisAsnGlnIleGlyGlySerLysGluSerAsnLys                              405410415                                                                     ProAlaMetAlaHisArgAspIleLysSerLysAsnIleMetValLys                              420425430                                                                     AsnAspLeuThrCysAlaIleGlyAspLeuGlyLeuSerLeuSerLys                              435440445                                                                     ProGluAspAlaAlaSerAspIleIleAlaAsnGluAsnTyrLysCys                              450455460                                                                     GlyThrValArgTyrLeuAlaProGluIleLeuAsnSerThrMetGln                              465470475480                                                                  PheThrValPheGluSerTyrGlnCysAlaAspValTyrSerPheSer                              485490495                                                                     LeuValMetTrpGluThrLeuCysArgCysGluAspGlyAspValLeu                              500505510                                                                     ProArgGluAlaAlaThrValIleProTyrIleGluTrpThrAspArg                              515520525                                                                     AspProGlnAspAlaGlnMetPheAspValValCysThrArgArgLeu                              530535540                                                                     ArgProThrGluAsnProLeuTrpLysAspHisProGluMetLysHis                              545550555560                                                                  IleMetGluIleIleLysThrCysTrpAsnGlyAsnProSerAlaArg                              565570575                                                                     PheThrSerTyrIleCysArgLysArgMetAspGluArgGlnGlnLeu                              580585590                                                                     LeuLeuAspLysLysAlaLysAlaValAlaGlnThrAlaGlyValThr                              595600605                                                                     ValGlnAspArgLysIleLeuGlyProGlnLysProLysAspGluSer                              610615620                                                                     ProAlaAsnGlyAlaProArgIleValGlnLysGluIleAspArgGlu                              625630635640                                                                  AspGluGlnGluAsnTrpArgGluThrAlaLysThrProAsnGlyHis                              645650655                                                                     IleSerSerAsnAspAspSerSerArgProLeuLeuGly                                       660665                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 565 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..565                                                          (D) OTHER INFORMATION: /note= "TGF-B TYPE II RECEPTOR"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetGlyArgGlyLeuLeuArgGlyLeuTrpProLeuHisIleValLeu                              151015                                                                        TrpThrArgIleAlaSerThrIleProProHisValGlnLysSerVal                              202530                                                                        AsnAsnAspMetIleValThrAspAsnAsnGlyAlaValLysPhePro                              354045                                                                        GlnLeuCysLysPheCysAspValArgPheSerThrCysAspAsnGln                              505560                                                                        LysSerCysMetSerAsnCysSerIleThrSerIleCysGluLysPro                              65707580                                                                      GlnGluValCysValAlaValTrpArgLysAsnAspGluAsnIleThr                              859095                                                                        LeuGluThrValCysHisAspProLysLeuProTyrHisAspPheIle                              100105110                                                                     LeuGluAspAlaAlaSerProLysCysIleMetLysGluLysLysLys                              115120125                                                                     ProGlyGluThrPhePheMetCysSerCysSerSerAspGluCysAsn                              130135140                                                                     AspAsnIleIlePheSerGluGluTyrAsnThrSerAsnProAspLeu                              145150155160                                                                  LeuLeuValIlePheGlnValThrGlyIleSerLeuLeuProProLeu                              165170175                                                                     GlyValAlaIleSerValIleIleIlePheTyrCysTyrArgValAsn                              180185190                                                                     ArgGlnGlnLysLeuSerSerThrTrpGluThrGlyLysThrArgLys                              195200205                                                                     LeuMetGluPheSerGluHisCysAlaIleIleLeuGluAspAspArg                              210215220                                                                     SerAspIleSerSerThrCysAlaAsnAsnIleAsnHisAsnThrGlu                              225230235240                                                                  LeuLeuProIleGluLeuAspThrLeuValGlyLysGlyArgPheAla                              245250255                                                                     GluValTyrLysAlaLysLeuLysGlnAsnThrSerGluGlnPheGlu                              260265270                                                                     ThrValAlaValLysIlePheProTyrGluGluTyrAlaSerTrpLys                              275280285                                                                     AspArgLysAspIlePheSerAspIleAsnLeuLysHisGluAsnIle                              290295300                                                                     LeuGlnPheLeuThrAlaGluGluArgLysThrGluLeuGlyLysGln                              305310315320                                                                  TyrTrpLeuIleThrAlaPheHisAlaLysGlyAsnLeuGlnGluTyr                              325330335                                                                     LeuThrArgHisValIleSerTrpGluAspLeuArgAsnValGlySer                              340345350                                                                     SerLeuAlaArgGlyLeuSerHisLeuHisSerAspHisThrProCys                              355360365                                                                     GlyArgProLysMetProIleValHisArgAspLeuLysSerSerAsn                              370375380                                                                     IleLeuValLysAsnAspLeuThrCysCysLeuCysAspPheGlyLeu                              385390395400                                                                  SerLeuArgLeuGlyProTyrSerSerValAspAspLeuAlaAsnSer                              405410415                                                                     GlyGlnValGlyThrAlaArgTyrMetAlaProGluValLeuGluSer                              420425430                                                                     ArgMetAsnLeuGluAsnAlaGluSerPheLysGlnThrAspValTyr                              435440445                                                                     SerMetAlaLeuValLeuTrpGluMetThrSerArgCysAsnAlaVal                              450455460                                                                     GlyGluValLysAspTyrGluProProPheGlySerLysValArgAsp                              465470475480                                                                  ProValValGluSerMetLysAspAsnValLeuArgAspArgGlyThr                              485490495                                                                     ArgAsnSerSerPheTrpLeuAsnHisGlnGlyIleGlnMetValCys                              500505510                                                                     GluThrLeuThrGluCysTrpAspHisAspProGluAlaArgLeuThr                              515520525                                                                     AlaGlnCysValAlaGluArgPheSerGluLeuGluHisLeuAspArg                              530535540                                                                     LeuSerGlyArgSerCysSerGluGluLysIleProGluAspGlySer                              545550555560                                                                  LeuAsnThrThrLys                                                               565                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 513 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..513                                                          (D) OTHER INFORMATION: /note= "MOUSE ACTIVIN RECEPTOR"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetGlyAlaAlaAlaLysLeuAlaPheAlaValPheLeuIleSerCys                              151015                                                                        SerSerGlyAlaIleLeuGlyArgSerGluThrGlnGluCysLeuPhe                              202530                                                                        PheAsnAlaAsnTrpGluArgAspArgThrAsnGlnThrGlyValGlu                              354045                                                                        ProCysTyrGlyAspLysAspLysArgArgHisCysPheAlaThrTrp                              505560                                                                        LysAsnIleSerGlySerIleGluIleValLysGlnGlyCysTrpLeu                              65707580                                                                      AspAspIleAsnCysTyrAspArgThrAspCysIleGluLysLysAsp                              859095                                                                        SerProGluValTyrPheCysCysCysGluGlyAsnMetCysAsnGlu                              100105110                                                                     LysPheSerTyrPheProGluMetGluValThrGlnProThrSerAsn                              115120125                                                                     ProValThrProLysProProTyrTyrAsnIleLeuLeuTyrSerLeu                              130135140                                                                     ValProLeuMetLeuIleAlaGlyIleValIleCysAlaPheTrpVal                              145150155160                                                                  TyrArgHisHisLysMetAlaTyrProProValLeuValProThrGln                              165170175                                                                     AspProGlyProProProProSerProLeuLeuGlyLeuLysProLeu                              180185190                                                                     GlnLeuLeuGluValLysAlaArgGlyArgPheGlyCysValTrpLys                              195200205                                                                     AlaGlnLeuLeuAsnGluTyrValAlaValLysIlePheProIleGln                              210215220                                                                     AspLysGlnSerTrpGlnAsnGluTyrGluValTyrSerLeuProGly                              225230235240                                                                  MetLysHisGluAsnIleLeuGlnPheIleGlyAlaGluLysArgGly                              245250255                                                                     ThrSerValAspValAspLeuTrpLeuIleThrAlaPheHisGluLys                              260265270                                                                     GlySerLeuSerAspPheLeuLysAlaAsnValValSerTrpAsnGlu                              275280285                                                                     LeuCysHisIleAlaGluThrMetAlaArgGlyLeuAlaTyrLeuHis                              290295300                                                                     GluAspIleProGlyLeuLysAspGlyHisLysProAlaIleSerHis                              305310315320                                                                  ArgAspIleLysSerLysAsnValLeuLeuLysAsnAsnLeuThrAla                              325330335                                                                     CysIleAlaAspPheGlyLeuAlaLeuLysPheGluAlaGlyLysSer                              340345350                                                                     AlaGlyAspThrHisGlyGlnValGlyThrArgArgTyrMetAlaPro                              355360365                                                                     GluValLeuGluGlyAlaIleAsnPheGlnArgAspAlaPheLeuArg                              370375380                                                                     IleAspMetTyrAlaMetGlyLeuValLeuTrpGluLeuAlaSerArg                              385390395400                                                                  CysThrAlaAlaAspGlyProValAspGluTyrMetLeuProPheGlu                              405410415                                                                     GluGluIleGlyGlnHisProSerLeuGluAspMetGlnGluValVal                              420425430                                                                     ValHisLysLysLysArgProValLeuArgAspTyrTrpGlnLysHis                              435440445                                                                     AlaGlyMetAlaMetLeuCysGluThrIleGluGluCysTrpAspHis                              450455460                                                                     AspAlaGluAlaArgLeuSerAlaGlyCysValGlyGluArgIleThr                              465470475480                                                                  GlnMetGlnArgLeuThrAsnIleIleThrThrGluAspIleValThr                              485490495                                                                     ValValThrMetValThrAsnValAspPheProProLysGluSerSer                              500505510                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 513 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..513                                                          (D) OTHER INFORMATION: /note= "RAT ACTIVIN TYPE II                            RECEPTOR"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetThrAlaProTrpAlaAlaLeuAlaLeuLeuTrpGlySerLeuCys                              151015                                                                        AlaGlySerGlyArgGlyGluAlaGluThrArgGluCysIleTyrTyr                              202530                                                                        AsnAlaAsnTrpGluLeuGluArgThrAsnGlnSerGlyLeuGluArg                              354045                                                                        CysGluGlyGluGlnAspLysArgLeuHisCysTyrAlaSerTrpPro                              505560                                                                        AsnSerSerGlyThrIleGluLeuValLysLysGlyCysTrpLeuAsp                              65707580                                                                      AspPheAsnCysTyrAspArgGlnGluCysValAlaThrGluGluAsn                              859095                                                                        ProGlnValTyrPheCysCysCysGluGlyAsnPheCysAsnGluArg                              100105110                                                                     PheThrHisLeuProGluProGlyGlyProGluValThrTyrGluPro                              115120125                                                                     ProProThrAlaProThrLeuLeuThrValLeuAlaTyrSerLeuLeu                              130135140                                                                     ProIleGlyGlyLeuSerLeuIleValLeuLeuAlaPheTrpMetTyr                              145150155160                                                                  ArgHisArgLysProProTyrGlyHisValAspIleHisGluAspPro                              165170175                                                                     GlyProProProProSerProLeuValGlyLeuLysProLeuGlnLeu                              180185190                                                                     LeuGluIleLysAlaArgGlyArgPheGlyCysValTrpLysAlaGln                              195200205                                                                     LeuMetAsnAspPheValAlaValLysIlePheProLeuGlnAspLys                              210215220                                                                     GlnSerTrpGlnSerGluArgGluIlePheSerThrProGlyMetLys                              225230235240                                                                  HisGluAsnLeuLeuGlnPheIleAlaAlaGluLysArgGlyCysSer                              245250255                                                                     AsnLeuGluValGluLeuTrpLeuIleThrAlaPheHisAspLysGly                              260265270                                                                     SerLeuThrAspTyrLeuLysGlyAsnIleIleThrTrpAsnGluLeu                              275280285                                                                     CysHisValAlaGluThrMetSerArgGlyLeuSerTyrLeuHisGlu                              290295300                                                                     AspValProTrpCysArgGlyGluGlyHisLysProSerIleAlaHis                              305310315320                                                                  ArgAspPheLysSerLysAsnValLeuLeuLysSerAspLeuThrAla                              325330335                                                                     ValLeuAlaAspPheGlyLeuAlaValArgPheGluProGlyLysPro                              340345350                                                                     ProGlyAspThrHisGlyGlnValGlyThrArgArgTyrMetAlaPro                              355360365                                                                     GluValLeuGluGlyAlaIleAsnPheGlnArgAspAlaPheLeuArg                              370375380                                                                     IleAspMetTyrAlaMetGlyLeuValLeuTrpGluLeuValSerArg                              385390395400                                                                  CysLysAlaAlaAspGlyProValAspGluTyrMetLeuProPheGlu                              405410415                                                                     GluGluIleGlyGlnHisProSerLeuGluGluLeuGlnGluValVal                              420425430                                                                     ValHisLysLysMetArgProThrIleLysAspHisTrpLeuLysHis                              435440445                                                                     ProGlyLeuAlaGlnLeuCysValThrIleGluGluCysTrpAspHis                              450455460                                                                     AspAlaGluAlaArgLeuSerAlaGlyCysValGluGluArgValSer                              465470475480                                                                  LeuIleArgArgSerValAsnGlySerThrSerAspCysLeuValSer                              485490495                                                                     LeuValThrSerSerThrAsnValAspLeuLeuProLysGluSerSer                              500505510                                                                     Ile                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 536 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..536                                                          (D) OTHER INFORMATION: /note= "HUMAN ACTIVIN TYPE II                          RECEPTOR"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetThrAlaProTrpAlaAlaLeuAlaLeuLeuTrpGlySerLeuCys                              151015                                                                        AlaGlySerGlyArgGlyGluAlaGluThrArgGluCysIleTyrTyr                              202530                                                                        AsnAlaAsnTrpGluLeuGluArgThrAsnGlnSerGlyLeuGluArg                              354045                                                                        CysGluGlyGluGlnAspLysArgLeuHisCysTyrAlaSerTrpAla                              505560                                                                        AsnSerSerGlyThrIleGluLeuValLysLysGlyCysTrpLeuAsp                              65707580                                                                      AspPheAsnCysTyrAspArgGlnGluCysValAlaThrGluGluAsn                              859095                                                                        ProGlnValTyrPheCysCysCysGluGlyAsnPheCysAsnGluArg                              100105110                                                                     PheThrHisLeuProGluProGlyGlyProGluValThrTyrGluPro                              115120125                                                                     ProProThrAlaProThrLeuLeuThrValLeuAlaTyrSerLeuLeu                              130135140                                                                     ProIleGlyGlyLeuSerLeuIleValLeuLeuAlaPheTrpMetTyr                              145150155160                                                                  ArgHisArgLysProProTyrGlyHisValAspIleHisGluValArg                              165170175                                                                     GlnCysGlnArgTrpAlaGlyArgArgAspGlyCysAlaAspSerPhe                              180185190                                                                     LysProLeuProPheGlnAspProGlyProProProProSerProLeu                              195200205                                                                     ValGlyLeuLysProLeuGlnLeuLeuGluIleLysAlaArgGlyArg                              210215220                                                                     PheGlyCysValTrpLysAlaGlnLeuMetAsnAspPheValAlaVal                              225230235240                                                                  LysIlePheProLeuGlnAspLysGlnSerTrpGlnSerGluArgGlu                              245250255                                                                     IlePheSerThrProGlyMetLysHisGluAsnLeuLeuGlnPheIle                              260265270                                                                     AlaAlaGluLysArgGlySerAsnLeuGluValGluLeuTrpLeuIle                              275280285                                                                     ThrAlaPheHisAspLysGlySerLeuThrAspTyrLeuLysGlyAsn                              290295300                                                                     IleIleThrTrpAsnGluLeuCysHisValAlaGluThrMetSerArg                              305310315320                                                                  GlyLeuSerTyrLeuHisGluAspValProTrpCysArgGlyGluGly                              325330335                                                                     HisLysProSerIleAlaHisArgAspPheLysSerLysAsnValLeu                              340345350                                                                     LeuLysSerAspLeuThrAlaValLeuAlaAspPheGlyLeuAlaVal                              355360365                                                                     ArgPheGluProGlyLysProProGlyAspThrHisGlyGlnValGly                              370375380                                                                     ThrArgArgTyrMetAlaProGluValLeuGluGlyAlaIleAsnPhe                              385390395400                                                                  GlnArgAspAlaPheLeuArgIleAspMetTyrAlaMetGlyLeuVal                              405410415                                                                     LeuTrpGluLeuValSerArgCysLysAlaAlaAspGlyProValAsp                              420425430                                                                     GluTyrMetLeuProPheGluGluGluIleGlyGlnHisProSerLeu                              435440445                                                                     GluGluLeuGlnGluValValValHisLysLysMetArgProThrIle                              450455460                                                                     LysAspHisTrpLeuLysHisProGlyLeuAlaGlnLeuCysValThr                              465470475480                                                                  IleGluGluCysTrpAspHisAspAlaGluAlaArgLeuSerAlaGly                              485490495                                                                     CysValGluGluArgValSerLeuIleArgArgSerValAsnGlyThr                              500505510                                                                     ThrSerAspCysLeuValSerLeuValThrSerValThrAsnValAsp                              515520525                                                                     LeuLeuProLysGluSerSerIle                                                      530535                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid fragment comprising:(a)the nucleotide sequence defined by nucleotide bases 451-819 of SEQ IDNO: 1; or (b) the nucleotide sequence encoding amino acids 25-147 of SEQID NO: 2 or conservative amino acid substitutions thereof.
 2. Anisolated nucleic acid fragment comprising:(a) the nucleotide sequencedefined by nucleotide bases 1003-1854 of SEQ ID NO: 1; or (b) thenucleotide sequence encoding amino acids 209-492 of SEQ ID NO: 2 orconservative amino acid substitutions thereof.
 3. An isolated nucleicacid fragment comprising:(a) the coding nucleotide sequence defined bySEQ ID NO: 1; or (b) the nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 2 or conservative amino acid substitutionsthereof.